Targets for therapeutic intervention identified in the mitochondrial proteome

STATEMENT REGARDING SEQUENCE LISTING SUBMITTED ON CD-ROM

[0002] The Sequence Listing associated with this application is provided on CD-ROM in lieu of a paper copy, and is hereby incorporated by reference into the specification. Three CD-ROMs are provided, containing identical copies of the sequence listing: CD-ROM No. 1 is labeled COPY 1, contains the file 465.app.txt which is 14.4 MB and created on Apr. 4, 2003; CD-ROM No.2 is labeled COPY 2, contains the file 465.app.txt which is 14.4 MB and created on Apr. 4, 2003; CD-ROM No. 3 is labeled CRF, contains the file 465.app.txt which is 14.4 MB and created on Apr. 4, 2003.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to compositions and methods for identifying mitochondrial proteins that are useful as targets for therapeutic intervention in treating diseases associated with altered mitochondrial function. More specifically, the invention is directed to proteomic profiling of proteins and polypeptides of mitochondria and to uses of mitochondrial polypeptides in screening assays for, and as targets of, therapeutic agents.

[0005] 2. Description of the Related Art

[0006] Mitochondria are the complex subcellular organelles that manufacture bioenergetically essential adenosine triphosphate (ATP) by oxidative phosphorylation, and that promote direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes, including aerobic respiration and intracellular calcium regulation. For example, mitochondria provide the subcellular site for physiologically important processes such as the Krebs cycle, the urea cycle, fatty acid β-oxidation, and heme synthesis. Mitochondria also participate in mechanisms of apoptosis, or programmed cell death (e.g., Newmeyer et al., Cell 79:353-364,1994; Liu et al., Cell 86:147-157, 1996), which is apparently required for, inter alia, normal development of the nervous system and proper functioning of the immune system.

[0007] Functional mitochondria contain gene products encoded by mitochondrial genes situated in mitochondrial DNA (mtDNA) and by extramitochondrial (e.g., nuclear) genes not situated in the circular mitochondrial genome. While it has been estimated that a functional human mitochondrion contains on the order of 1,000-1,500 distinct proteins (Lopez et al., 2000 Electrophoresis 21:3427; Scheffler, I. E., Mitochondria, 1999 Wiley-Liss, Inc., New York; Rabilloud et al., 1998 Electrophoresis 19:1006; Scheffleretal., 2001 Mitochondrion 1:161; Schatz, G., 1995 Biochem. Biophys. Acta Mol. Basis Dis. 1271:123), the 16.5 kb mtDNA encodes 22 tRNAs, two ribosomal RNAs (12s and 16s rRNA) and only 13 polypeptides, which are enzymes of the electron transport chain (ETC), the elaborate multi-subunit complex mitochondrial assembly where, for example, respiratory oxidative phosphorylation takes place. (See, e.g., Wallace et al., in Mitochondria & Free Radicals in Neurodegenerative Diseases, M. F. Beal, N. Howell and I. BodisWollner, eds., 1997 Wiley-Liss, Inc., New York, pp. 283-307, and references cited therein; see also, e.g., Scheffler, I. E., Mitochondria, 1999Wiley-Liss, Inc., New York.) Mitochondrial DNA thus includes gene sequences encoding seven subunits of NADH dehydrogenase, also known as ETC Complex I (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6); one subunit of ETC Complex III (ubiquinol: cytochrome c oxidoreductase, Cytb); three cytochrome c oxidase (ETC Complex IV) subunits (COX1, COX2 and COX3); and two proton-translocating ATP synthase (Complex V) subunits (ATPase6 and ATPase8). All other mitochondrial constituent polypeptides are presumed to be encoded by genes of the extramitochondrial genome, and the number and identities of a large number of these polypeptides remain unknown. Accordingly, for most of the estimated 25,000-40,000 proteins encoded by the human nuclear genome (Venter et al., 2001 Science 291:1304; Lander et al., 2001 Nature 409:860) little is known regarding subcellular localization, for example, which proteins may be molecular components of mitochondria.

[0008] Mitochondria contain an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix (for review, see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s.) The cristae, originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane by open, circular (30 nm diameter) junctions (Perkins et al., 1997, JI. of Struct. Biol. 119:260). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (greater than about 10 kD) molecules.

[0009] Four of the five multisubunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane. The remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical membrane potential of approximately 220 mV referred to as the “protonmotive force” (PMF). The PMF, which is often represented by the notation Δp, corresponds to the sum of the electric potential (Δψm) and the pH differential (ΔpH) across the inner membrane according to the equation

Δp=Δωm−ZΔpH

[0010] wherein Z stands for −2.303 RT/F. The value of Z is −59 at 25° C. when Δp and Δψm are expressed in mV and ΔpH is expressed in pH units (see, e.g., Ernster et al., J. Cell Biol. 91:227s, 1981 and references cited therein).

[0011] Δψm provides the energy for phosphorylation of adenosine diphosphate (ADP) to yield ATP by ETC Complex V, a process that is coupled stoichiometrically with transport of a proton into the matrix. Δψm is also the driving force for the influx of cytosolic Ca2+ into the mitochondrion. Under normal metabolic conditions, the inner membrane is impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the sole means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during mitochondrial permeability transition (MPT) that accompanies certain diseases associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration. During MPT, Δψm collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca2+, Na+, K+ and H+) and large (e.g., proteins).

[0012] A number of diseases, disorders or conditions, including degenerative diseases, are thought to be caused by, or are associated with, alterations in mitochondrial function as provided herein. These disorders include Alzheimer's Disease (AD), diabetes mellitus, Parkinson's Disease (PD), Huntington's disease, Freidreich's ataxia, atherosclerosis, hypertension, ischemia-reperfusion injury, osteoarthritis, inflammatory diseases, amyotrophic lateral sclerosis (ALS), Wilson disease, autosomal recessive hereditary spastic paraplegia, Leigh syndrome, benign and fatal infantile myopathies, multiple sclerosis, dystonia, Leber's hereditary optic neuropathy, schizophrenia, cancer; psoriasis; Down's syndrome, hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myodegenerative disorders such as “mitochondrial encephalopathy, lactic acidosis, and stroke” (MELAS), and “myoclonic epilepsy ragged red fiber syndrome” (MERRF), as well as other mitochondrial respiratory chain diseases (reviewed in Chinnery et al., 1999 J. Med. Genet. 36:425; see also references cited therein). Diseases associated with altered mitochondrial function thus include these and other diseases in which one or more levels of an indicator of altered mitochondrial function differ in a statistically significant manner from the corresponding indicator levels found in clinically normal subjects known to be free of a presence or risk of such disease. Other diseases involving altered metabolism or respiration within cells may also be regarded as diseases associated with altered mitochondrial function, for example, those in which free radicals such as reactive oxygen species (ROS) contribute to pathogenesis. Certain diseases associated with altered mitochondrial function appear to involve states of insufficient apoptosis (e.g., cancer and autoimmune diseases) or excessive levels of apoptosis (e.g., stroke and neurodegeneration). For a general review of apoptosis, and the role of mitochondria therein, see, e.g., Green and Reed, Science 281:1309-1312, 1998; Green, Cell 94:695-698, 1998 and Kromer, Nature Medicine 3:614-620, 1997. The extensive list of additional diseases associated with altered mitochondrial function continues to expand as aberrant mitochondrial or mitonuclear activities are implicated in particular disease processes.

[0013] For instance, free radical production in biological systems is known to result in the generation of reactive species that can chemically modify molecular components of cells and tissues. Such modifications can alter or disrupt structural and/or functional properties of these molecules, leading to compromised cellular activity and tissue damage. Mitochondria are a primary source of free radicals in biological systems (see, e.g., Murphy et al., 1998 in Mitochondria and Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Woliner, Eds., Wiley-Liss, New York, pp. 159-186 and references cited therein), and altered mitochondrial function, such as failure at any step of the mitochondrial electron transport chain (ETC), may also lead to the generation of highly reactive free radicals. Thus, free radicals generated in biological systems, including free radicals resulting from altered mitochondrial function or from extramitochondrial sources, include reactive oxygen species (ROS), for example, superoxide, peroxynitrite and hydroxyl radicals, and potentially other reactive species that may be toxic to cells. Diseases associated with altered mitochondrial function therefore include disorders in which free radicals contribute to pathogenesis at the molecular level (see, e.g., Halliwell B. and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, 1989 Clarendon Press, Oxford, UK).

[0014] A particularly prevalent example of a disease associated with altered mitochondrial function is type 2 diabetes mellitus, or “late onset” diabetes, a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing type 2 diabetes mellitus (“type 2 DM”) is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.

[0015] At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that type 2 DM may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from impaired glucose tolerance (IGT). Following a glucose load, ciruculating glucose concentrations in IGT patients rise to higher levels, and return to baseline levels more slowly, than in unaffected individuals. A small percentage of IGT individuals (5-10%) progress to non-insulin dependent diabetes (NIDDM) each year. This form of diabetes mellitus, type 2 DM, is associated with decreased release of insulin by pancreatic beta cells and a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies and blindness.

[0016] Despite intense effort, nuclear genes that segregate with diabetes mellitus are rare and include, for example, mutations in the insulin gene, the insulin receptor gene and the glucokinase gene. By comparison, although a number of altered mitochondrial genes that segregate with diabetes mellitus have been reported (see generally e.g., PCT/US95/04063), relationships amongst mitochondrial and extramitochondrial factors that contribute to cellular respiratory and/or metabolic activities as they pertain to diabetes remain poorly understood.

[0017] Current pharmacological therapies for type 2 DM include injected insulin, and oral agents that are designed to lower blood glucose levels. Currently available oral agents include (i) the sulfonylureas, which act by enhancing the sensitivity of the pancreatic beta cell to glucose, thereby increasing insulin secretion in response to a given glucose load; (ii) the biguanides, which improve glucose disposal rates and inhibit hepatic glucose output; (iii) the thiazolidinediones, which improve peripheral insulin sensitivity through interaction with nuclear peroxisome proliferator-activated receptors (PPAR, see, e.g., Spiegelman, 1998 Diabetes 47:507-514; Schoonjans et al., 1997 Curr. Opin. Lipidol. 8:159-166; Staels et al., 1997 Biochimie 79:95-99), (iv) repaglinide, which enhances insulin secretion through interaction with ATP-dependent potassium channels; and (v) acarbose, which decreases intestinal absorption of carbohydrates. It is clear that none of the current pharmacological therapies corrects the underlying biochemical defect in type 2 DM. Neither do any of these. currently available treatments improve all of the physiological abnormalities in type 2 DM such as impaired insulin secretion, insulin resistance and/or excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.

[0018] Clearly there is a need for improved diagnostic methods for early detection of a risk for developing a disease associated with altered mitochondrial function, and for better therapeutics that are specifically targeted to correct biochemical and/or metabolic defects responsible for such disease, regardless of whether such a defect underlying altered mitochondrial function may have mitochondrial or extramitochondrial origins. The present invention provides compositions and methods related to identification of mitochondrial targets for therapeutic intervention in treating these diseases, and offers other related advantages.

BRIEF SUMMARY OF THE INVENTION

[0019] The present invention provides the identities of 3025 polypeptide sequences [SEQ ID NOS:1-3025] that are constituents of the human mitochondrial proteome. It is therefore an aspect of the present invention to provide a method for identifying a mitochondrial target for therapeutic intervention in treatment of a disease associated with altered mitochondrial function, comprising (a) determining a presence, in a biological sample from a subject known to have or suspected of having a disease associated with altered mitochondrial function, of at least one modified polypeptide, the modified polypeptide comprising at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025; and (b) correlating the modification with at least one disease associated with altered mitochondrial function, and therefrom identifying a mitochondrial target for therapeutic intervention.

[0020] In certain embodiments the modified polypeptide exhibits altered biological activity. In certain embodiments the biological sample is selected from the group consisting of blood, skin, skeletal muscle, liver and cartilage. In certain embodiments the disease associated with altered mitochondrial function is Alzheimer's disease, diabetes mellitus, Parkinson's disease, Huntington's disease, osteoarthritis, dystonia, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS), myoclonic epilepsy ragged red fiber syndrome (MERRF) or cancer. In certain embodiments the modification is an amino acid substitution, an amino acid insertion, an amino acid deletion, a posttranslational modification or an altered expression level, and in certain further embodiments the posttranslational modification is glycosylation, phosphorylation, nitration, nitrosylation, amidation, fatty acylation or oxidative modification, including, for example, oxidative post-translational modification of tryptophan residues.

[0021] In certain other embodiments the present invention provides a method of identifying an agent for treating a disease associated with altered mitochondrial function, comprising (a) contacting a candidate agent with a biological sample from a subject having a disease associated with altered mitochondrial function, wherein the sample comprises at least one polypeptide that exhibits altered biological activity which accompanies the disease and wherein the polypeptide is (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, or (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025; and (b) determining an increase or decrease in the altered biological activity of the polypeptide in the presence of the candidate agent relative to the level of the altered biological activity in the absence of the candidate agent, and therefrom identifying an agent for treating a disease associated with altered mitochondrial function.

[0022] In certain embodiments the altered biological activity is an indicator of altered mitochondrial function that is ATP biosynthesis (e.g., an ATP biosynthesis factor), oxidative phosphorylation, mitochondrial calcium uptake, mitochondrial calcium release, maintenance of inner mitochondrial membrane potential, mitochondrial permeability transition, ETC-mediated electron transport or mitochondrial intermembrane space protein release. In certain other embodiments the sample is a cell, a mitochondria enriched sample, an isolated mitochondrion or a submitochondrial particle. In certain embodiments the disease associated with-altered mitochondrial function is Alzheimer's disease, diabetes mellitus, Parkinson's disease, Huntington's disease, osteoarthritis, dystonia, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS), myoclonic epilepsy ragged red fiber syndrome (MERRF) or cancer.

[0023] According to certain other embodiments there is provided by the present invention a method of treating a disease associated with altered mitochondrial function comprising administering to a subject in need thereof an agent that compensates for at least one biological activity of a polypeptide that exhibits altered biological activity which accompanies the disease, wherein the polypeptide is (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, or (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025. In another embodiment the invention provides a method for identifying a risk for having or a presence of a disease associated with altered mitochondrial function, comprising (a) determining a presence, in a biological sample from a subject suspected of having a disease associated with altered mitochondrial function, of at least one modified polypeptide, the modified polypeptide comprising at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, wherein the modification correlates with at least one disease associated with altered mitochondrial function, and therefrom identifying a risk for or presence of disease.

[0024] Certain other embodiments of the invention provide a method of identifying an agent for treating a disease associated with altered mitochondrial function, comprising (a) contacting a candidate agent with an isolated polypeptide that exhibits altered biological activity which accompanies a disease associated with altered mitochondrial function, wherein the polypeptide is selected from the group consisting of (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025 and (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025; and (b) determining an increase or decrease in the altered biological activity of the polypeptide in the presence of the candidate agent relative to the level of the altered biological activity in the absence of the candidate agent, and therefrom identifying an agent for treating a disease associated with altered mitochondrial function. In certain further embodiments the disease associated with altered mitochondrial function is Alzheimer's disease, diabetes mellitus, Parkinson's disease, Huntington's disease, osteoarthritis, dystonia, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS), myoclonic epilepsy ragged red fiber syndrome (MERRF), or cancer. In other further embodiments the isolated polypeptide is present in a preparation that is a submitochondrial particle, a proteoliposome or a mitochondrial protein fraction.

[0025] In another embodiment the invention provides a method of identifying an agent for treating a disease associated with altered mitochondrial function, comprising (a) administering a candidate agent to a subject having a disease associated with altered mitochondrial function; and (b) determining, in a first biological sample obtained from the subject prior to the step of administering the candidate agent and in a second biological sample obtained from the subject subsequent to the step of administering the candidate agent, wherein each of said first and second samples comprises at least one polypeptide that exhibits altered biological activity which accompanies said disease and wherein the polypeptide is selected from the group consisting of (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025 and (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, an increase or decrease in the altered biological activity of the polypeptide in the second sample relative to the level of the altered biological activity in the first sample, and therefrom identifying an agent for treating a disease associated with altered mitochondrial function. In a further embodiment, the altered biological activity is an indicator of altered mitochondrial function that is ATP biosynthesis, oxidative phosphorylation, calcium uptake, calcium release, maintenance of inner mitochondrial membrane potential, mitochondrial permeability transition, ETC-mediated electron transport or intermembrane space protein release. In another further embodiment the sample is a cell, a mitochondria enriched sample, an isolated mitochondrion or a submitochondrial particle. In certain other further embodiments, the disease associated with altered mitochondrial function is Alzheimer's disease, diabetes mellitus, Parkinson's disease, Huntington's disease, osteoarthritis, dystonia, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS), myoclonic epilepsy ragged red fiber syndrome (MERRF), or cancer.

[0026] These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions and are therefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]
FIG. 1 shows representative western immunoblot analysis (FIG. 1A) of indicated mitochondrial ETC proteins in sucrose density gradient fractionated isolated human heart mitochondria, following resolution of proteins by one-dimensional polyacrylamide gel electrophoresis (FIG. 1B).

[0028]
FIG. 2 shows a representative MALDI mass spectrum for a single band excised from a one-dimensional polyacrylamide gel following electrophoretic resolution of proteins from sucrose density gradient fractionated isolated human heart mitochondria. Peptides are from indicated mitochondrial proteins as follows: β=ATP synthase beta subunit, γ=ATP synthase gamma subunit, eCoA=enlyl-CoA hydratase, and vd=voltage dependent anion channel 1 (VDAC-1). (K=keratin.)

[0029]
FIG. 3 shows products of tryptophan oxidation in proteins.

[0030]
FIG. 4 shows MALDI-TOF mass spectrometry of two peptides from complex I subunit NDUFS4 displaying (A) tryptophan and (B) methionine oxidation. The samples were as follows (i) human heart mitochondria complex I (HHM individual #1) prepared by sucrose density gradient fractionation (SDG) and 1D electrophoresis; (ii) HHM individual #1 prepared by immunocapture and ID electrophoresis (iii) HHM individual #2 prepared by immunocapture and 1D electrophoresis; (iv) HHM individuals #3,4,5 (pooled) prepared by SDG and 1D electrophoresis; (v) bovine heart mitochondria (BHM animal #1) prepared by SDG and 1D electrophoresis; (vi) (BHM animal #2) prepared by SDG and 2D electrophoresis.

[0031]
FIG. 5 shows a comparison of the distribution of (a) tryptophan and (b) methionine oxidation for complex I subunit peptides.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides a method for identifying mitochondrial polypeptide targets for therapeutic intervention in the treatment of diseases associated with altered mitochondrial function, and a method for identifying agents for treating such diseases, as well as other related advantages.

[0033] The invention derives from characterization of the human heart mitochondrial proteome as described herein, to arrive at the surprising discovery and recognition for the first time that polypeptides having the amino acid sequences set forth in SEQ ID NOS:1-3025 are mitochondrial molecular components. This unexpected determination, that isolated human mitochondria comprise polypeptides having the amino acid sequences set forth in SEQ ID NOS:1-3025, is usefully combined with methods for determining the presence of a disease associated with altered mitochondrial function, and with methods for determining modification to, and altered biological activity of, a polypeptide, to provide targets for drug-screening assays and for therapeutic agents. According to certain embodiments, the invention relates to determination of at least one modified polypeptide that comprises a modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS:1-3025, and according to certain other embodiments the invention relates to determination of a profile comprising a plurality (e.g., two or more) of polypeptides having distinct amino acid sequences wherein at least one such polypeptide has one of the amino sequences set forth in SEQ ID NOS:1-3025, and has not been previously identified as a mitochondrial component.

[0034] Thus, it is an aspect of the present invention to provide a method for identifying a mitochondrial target for therapeutic intervention in treatment of a disease associated with altered mitochondrial function, comprising (a) determining a presence, in a biological sample from a subject known to have or suspected of having a disease associated with altered mitochondrial function, of at least one modified polypeptide, the modified polypeptide comprising at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025; and (b) correlating the modification with at least one disease associated with altered mitochondrial function, and therefrom identifying a mitochondrial target for therapeutic intervention.

[0035] Biological samples may comprise any tissue or cell preparation containing mitochondria. Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source. The subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortal, immortalized or immortalizable cell lines (e.g., capable of at least ten cell doublings in vitro), somatic cell hybrid or cytoplasmic hybrid “cybrid” cell lines (including mitochondrial cybrid cells having nuclear and mitochondrial DNAs of differing biological origins, see, e.g., U.S. Pat. No. 5,888,498 and International Publication No. WO 95/26793), differentiated or differentiatable cell lines, transformed cell lines and the like. In certain preferred embodiments of the invention, the subject or biological source may be suspected of having or being at risk for having a disease associated with altered mitochondrial function, including, for example, altered mitochondrial molecular composition or constitution, or oxidative modification of one or more mitochondrial proteins, and in certain preferred embodiments of the invention the subject or biological source may be known to be free of a risk or presence of such a disease. In certain other preferred embodiments a biological sample comprises a cybrid cell line having nuclear and mitochondrial DNAs of differing biological origins, which in certain embodiments may be a human cell, an immortal cell, a neuronal cell, a neuroblastoma or other transformed cell, for example, a SH-SY5Y human neuroblastoma cell. In certain other particularly preferred embodiments a biological sample comprises a sample readily obtained from a subject or biological source, such as blood, skin, skeletal muscle, liver or cartilage.

[0036] By way of background, mitochondria are comprised of “mitochondrial molecular components”, which may be any protein, polypeptide, peptide, amino acid, or derivative thereof; any lipid, fatty acid or the like, or derivative thereof; any carbohydrate, saccharide or the like or derivative thereof, any nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any other biological molecule that is a constituent of a mitochondrion, which may include molecules that are integral or stable components of mitochondrial structure, and may also include molecules that may transiently associate with mitochondria under certain conditions, for example, regulated intracellular events that involve mitochondria. In the most preferred embodiments, the present invention is directed to compositions and methods that relate to those mitochondrial molecular components that are mitochondrial polypeptides or proteins, although the invention need not be so limited.

[0037] In certain preferred embodiments of the present invention, a mitochondrial protein fraction is derived from the biological sample as provided herein. A protein fraction may be any preparation that contains at least one protein that is present in the sample and which may be obtained by processing a biological sample according to any biological and/or biochemical methods useful for isolating or otherwise separating a protein from its biological source. Those familiar with the art will be able to select an appropriate method depending on the biological starting material and other factors. Such methods may include, but need not be limited to, cell fractionation, density sedimentation, differential extraction, salt precipitation, ultrafiltration, gel filtration, ion-exchange chromatography, partition chromatography, hydrophobic chromatography, reversed-phase chromatography, one- and two-dimensional electrophoresis, affinity techniques or any other suitable separation method.

[0038] It will be noted that in certain particularly preferred embodiments of the present invention, at least one sample as described herein comprises a “mitochondria enriched” sample, which refers to a sample that comprises one or more mitochondria and that is substantially depleted (i.e., partially or fully depleted, where the degree of depletion of a given component can be quantified to show that its presence has been reduced in a statistically significant manner) of one or more non-mitochondrial marker proteins to the extent such markers can be removed from a preparation and are detectable, as described herein and known to the art. Thus, for example, cell fractionation techniques for the enrichment and detection of mitochondria, and/or biochemical markers characteristic of these and other defined organelles, may be used to determine that a particular subcellular fraction containing one or more detectable organelle-specific or organelle-associated markers or polypeptides, as provided herein, is substantially enriched in mitochondria (see, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s; see also, e.g., Rickwood et al., 1987, Mitochondria, a practical approach (Darley-Usmar, R., Wilson,, Ed.), IRL Press; Storrie and Madden, 1990 Methods in Enzymology 182, 203-225).

[0039] For example, and in certain preferred embodiments including methods for determining the presence in a biological sample of a mitochondrial target polypeptide for therapeutic intervention-or for screening a candidate agent for its ability to alter the biological activity of such a target, a mitochondrial molecular component such as any protein or polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS:1-3025 may be obtained from a preparation of isolated mitochondria and/or from a preparation of isolated submitochondrial particles (SMP). Techniques for isolating mitochondria and for preparing SMP are well known to the person having ordinary skill in the art and may include certain minor modifications as appropriate for the particular conditions selected (e.g., Smith, A. L., Meths. Enzymol. 10:81-86; Darley-Usman et al., (eds.), Mitochondria: A Practical Approach, IRL Press, Oxford, UK; Storrie et al., 1990 Meths. Enzymol. 182:203-255). Cell or tissue lysates, homogenates, extracts, suspensions, fractions or the like, or other preparations containing partially or fully purified mitochondrial molecular components such as mitochondrial proteins (e.g., MCA) may also be useful in these and related embodiments. According to certain other related embodiments, one or more isolated mitochondrial molecular components such as isolated targets for therapeutic intervention in the treatment of a disease associated with altered mitochondrial function may be present in membrane vesicles such as uni- or multilamellar membrane vesicles, or reconstituted into naturally derived or synthetic liposomes or proteoliposomes or similar membrane-bounded compartments, or the like, according to generally accepted methodologies (e.g., Jezek et al., 1990 J. Biol. Chem. 265:10522-10526).

[0040] Affinity techniques are particularly useful-in the context of the present invention, and may include any method that exploits a specific binding interaction with a mitochondrial protein or peptide to effect a separation. Other useful affinity techniques include immunological techniques for isolating specific proteins or peptides, which techniques rely on specific binding interaction between antibody combining sites for antigen and antigenic determinants present in the proteins or peptides. Immunological techniques include, but need not be limited to, immunoaffinity chromatography, immunoprecipitation, solid phase immunoadsorption or other immunoaffinity methods. See, for example, Scopes, R. K., Protein Purification: Principles and Practice, 1987, Springer-Verlag, NY; Weir, D. M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; Deutscher, M. P., Guide to Protein Purification, 1990, Methods in Enzymology Vol. 182, Academic Press, New York; and Hermanson, G. T. et al., Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., California; which are hereby incorporated by reference in their entireties, for details regarding techniques for isolating and characterizing proteins and peptides, including affinity techniques.

[0041] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For instance, a naturally occurring protein or peptide present in a living animal is not isolated, but the same protein or peptide, separated from some or all of the co-existing materials in the natural system, is isolated. Thus, for example, such proteins could be part of a multisubunit complex or a membrane vesicle, and/or such peptides could be part of a composition, and still be isolated in that such complex, vesicle or composition is not part of its natural environment.

[0042] “Biological activity” of a protein may be any detectable parameter that directly relates to a condition, process, pathway, dynamic structure, state or other activity involving the protein and that permits detection of altered protein function in a biological sample from a subject or biological source, or in a preparation of the protein isolated therefrom. The methods of the present invention thus pertain in part to such correlation where the protein having biological activity may be, for example, an enzyme, a structural protein, a receptor, a ligand, a membrane channel, a regulatory protein, a subunit, a complex component, a chaperone protein, a binding protein or a protein having a biological activity according to other criteria including those provided herein. Such activity may include the amount of a protein that is present, or the amount of a given protein's function that is detectable.

[0043] “Altered biological activity” of a protein may refer to any condition or state, including those that accompany a disease associated with altered mitochondrial function, for example, a disease or disorder characterized by altered (e.g., increased or decreased in a statistically significant manner relative to an appropriate control) mitochondrial molecular composition or constitution or by modification of a mitochondrial protein as provided herein (and in particular, e.g., a modification to a polypeptide that in its unmodified form comprises an amino acid sequence as set forth in any one of SEQ ID NOS:1-3025), where any structure or activity that is directly or indirectly related to a particular protein's function (or multiple functions) has been changed in a statistically significant manner relative to a control or standard.

[0044] Altered biological activity may have its origin in deletion, substitution or insertion of one or more amino acids in a mitochondrial protein; in posttranslational modification of a mitochondrial protein; in an altered expression level (e.g., a statistically significant increase or decrease in the amount present) of a mitochondrial protein; in oxidatively modified structures or oxidative events as well as in oxidation-independent structures or events, in direct interactions between mitochondrial and extramitochondrial genes and/or their gene products, or in structural or functional changes that occur as the result of interactions between intermediates that may be formed as the result of such interactions, including metabolites, catabolites, substrates, precursors, cofactors and the like. According to certain embodiments as provided herein, altered biological activity of a protein may also result from direct or indirect interaction of a biologically active protein with an introduced agent such as an agent for treating a disease associated with altered mitochondrial function as described herein, for example, a small molecule.

[0045] Additionally, altered biological activity of a mitochondrial protein (including proteins having any amino acid sequence set forth in SEQ ID NOS:1-3025 or modified forms of such proteins as provided herein) may result in altered respiratory, metabolic or other biochemical or biophysical activity in some or all cells of a biological source having a disease associated with altered mitochondrial function. As non-limiting examples, markedly impaired ETC activity may be related to altered biological activity of at least one protein, as may be generation of increased free radicals such as reactive oxygen species (ROS) or defective oxidative phosphorylation. As further examples, altered mitochondrial membrane potential, induction of apoptotic pathways and formation of a typical chemical and biochemical crosslinked species within a cell, whether by enzymatic or non-enzymatic mechanisms, may all be regarded as indicative of altered protein biological activity. Non-limiting examples of altered protein biological activity are described in greater detail below.

[0046] Thus, by way of non-limiting examples, coordinated replication of nuclear and mitochondrial DNA (reviewed in Clayton, D. A., 1992, Int. Rev. Cytol. 141, 217-232; and Shadel and Clayton, 1997, Annu. Rev. Biochem. 66, 409-435), or mitochondrial DNA transcription and RNA processing (Shadel and Clayton, 1996, Methods Enzymol. 264,149-158; Micol et al., 1996, Methods Enzymol. 264,158-173) both incompletely understood processes involving a large number of mitochondrial and extramitochondrial proteins, may be altered mitochondrial functions in certain diseases associated with altered mitochondrial function as provided herein. According to these examples, the disclosure herein—that polypeptides such as those listed in Table 2 alongside the functional classifications such as “carrier”, “DNA synthesis”, “nucleotide metabolism”, “transcription” and “transport”, are mitochondrial components—provides targets for therapeutic intervention in such diseases. In like manner, the disclosure herein that other polypeptides having amino acid sequences as set forth in SEQ ID NOS:1-3025 are mitochondrial components also identifies these proteins as targets for therapeutic intervention in a disease associated with altered mitochondrial function. Moreover, functional classifications of these proteins as recited in Tables 1 and 2 and in the GenBank annotations cited therein (which are incorporated by-reference) provides further guidance to those familiar with the art regarding how readily and without undue experimentation to select a biological activity for interrogation, to determine whether such activity is altered in a sample according to art accepted methodologies.

[0047] According to certain embodiments of the invention, a mitochondrial polypeptide is isolated from a biological sample following exposure of the sample to a “biological stimulus”, which may include any naturally occurring or artificial (including recombinant) compound that is capable of inducing altered biological activity of a mitochondrial molecular component which is, in preferred embodiments, a mitochondrial polypeptide. Thus, a biological stimulus may be employed, according to certain of the subject invention methods, to effect a perturbation of the biological status of a cell in a manner that alters biological activity of a mitochondrial polypeptide, such that the altered activity can be detected using any methodology described or referred to herein or known to the art, for example, according to the mass spectrometric fingerprinting methods described herein and in the cited references. Non-limiting examples of biological stimuli include antibodies, hormones, cytokines, chemokines, biologically active polypeptides and peptides and other soluble mediators, apoptogens, signal transduction agents, small molecules, cations and ionophores, physical and chemical stressors, and the like.

[0048] The polypeptides of the present invention are preferably provided in an isolated form, and in certain preferred embodiments are purified to homogeneity. The terms “fragment,” “derivative” and “analog” when referring to mitochondrial proteins such as polypeptides identified herein as mitochondrial components and having amino acid sequences as set forth in at least one of SEQ ID NOS:1-3025, or when referring to modified polypeptides that comprise at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS:1-3025 as provided herein, refers to any polypeptide or protein that retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active polypeptide.

[0049] The polypeptide (e.g., a human mitochondrial protein or polypeptide having an amino acid sequence set forth in SEQ ID NOS:1-3025) of the present invention may be a naturally occurring, a recombinant polypeptide or a synthetic polypeptide, and is preferably an isolated, naturally occurring polypeptide. Modified polypeptides according to the present invention comprise at least one modification (e.g., a structural change that occurs with statistical significance in a disease associated with altered mitochondrial function) to a protein or polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS:1-3025. The protein or polypeptide may therefore be an unmodified polypeptide or may be a polypeptide that has been posttranslationally modified, for example by glycosylation (e.g., N-linked glycosylation via asparagines residues, or O-linked glycoslyation via serine or threonine residues or post-biosynthetic glycation, etc.), phosphorylation, oxidation or oxidative modification, nitration, nitrosylation, amidation, fatty acylation including glycosylphosphatidylinositol anchor modification or the like, phospholipase cleavage such as phosphatidylinositol-specific phospholipase c mediated hydrolysis or the like, protease cleavage, dephosphorylation or any other type of protein posttranslational modification such as a modification involving formation or cleavage of a covalent chemical bond, although the invention need not be so limited and also contemplates non-covalent associations of proteins with other biomolecules (e.g., lipoproteins, metalloproteins, etc.). Methods for determining the presence of such modifications are well known in the art (e.g., Scopes, R. K., Protein Purification: Principles and Practice, 1987, Springer-Verlag, NY; Angeletti, Ed., Techniques in Protein Chemistry III, Academic Press, Inc., New York, 1993; Baynes et al., 1991 Diabetes 40:405; Baynes et al., 1999 Diabetes 48:1; Yamakura et al., 1998 J. Biol. Chem. 273:14085; MacMillan et al., 1998 Biochem. 37:1613; see also PCT/US01/14066).

[0050] A fragment, derivative or analog of a mitochondrial molecular component polypeptide or protein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, which may include a posttranslational modification or an adduct (e.g., an oxidative adduct), or (iii) one in which one or more of the amino acid residues are deleted, or (iv) one in which additional amino acids are fused to the polypeptide, including a signal sequence, a leader sequence or a proprotein sequence or the like, and also including additional peptide or non-peptide moieties that may be added to proteins such as ubiquitin, glutathione, thioredoxin and the like. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

[0051] The polypeptides of the present invention include mitochondrial polypeptides and proteins having amino acid sequences that are identical or similar to sequences known in the art. As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.

[0052] As described herein, isolation of a mitochondrial polypeptide component such as a mitochondrial molecular component with which an agent identified according to the methods of the invention interacts refers to physical separation of such a complex from its biological source, and may be accomplished by any of a number of well known techniques including but not limited to those described herein, and in the cited references. Without wishing to be bound by theory, a compound that “binds a mitochondrial component” can be any discrete molecule, agent compound, composition of matter or the like that may, but need not, directly bind to a mitochondrial molecular component, and may in the alternative bind indirectly to a mitochondrial molecular component by interacting with one or more additional components that bind to a mitochondrial molecular component. These or other mechanisms by which a compound may bind to and/or associate with a mitochondrial molecular component are within the scope of the claimed methods. Binding to a mitochondrial component may under certain conditions result in altered biological activity of the mitochondrial component.

[0053] According to certain preferred embodiments of the present invention, proteins and polypeptides comprising one or more of the amino acid sequences set forth in SEQ ID NOS:1-3025, which include polypeptides not previously known to be mitochondrial components, may be targets for drug screening and/or for therapeutic intervention. A “target” refers to a biochemical entity involved in a biological process, typically a protein that plays a useful role in the physiology or biology of a subject or biological source. A therapeutic composition or compound may bind to, alter the conformation of, impair or enhance the activity of or otherwise influence a target to alter (e.g., increase or decrease in a statistically significant manner relative to an appropriate untreated control) its function. As used herein, targets can include, but need not be limited to, proteins having a mitochondrial function classification as summarized in Table 2 and as described in greater detail below.

[0054] For example, targets may include proteins that are components of, or that associate with, mitochondrial ETC complexes, Krebs cycle or TCA cycle components including any molecules functionally linked (e.g., as substrates, cofactors, intermediates, biochemical donor or acceptor species, or the like) to such components, transport protein or carrier protein assemblies, factors or complexes involved in DNA (including mtDNA) replication or transcription or in translation of mRNA, cellular receptors, G-proteins or G-protein coupled receptors, kinases, phosphatases, ion channels, lipases, phosholipases, nuclear receptors and factors, intracellular structures, components of signal transduction and apoptotic pathways, and the like.

[0055] Methods for identifying a mitochondrial target (e.g., a pharmaceutical target such as a target for therapeutic intervention in a disease associated with altered mitochondrial function as provided herein, for instance, diabetes mellitus, a neurodegenerative disease, a disease associated with inappropriate cell proliferation or cell survival, or a cardiovascular condition) include providing a compound that modulates expression level, structure and/or activity of a particular mitochondrial protein (e.g., a component of the human mitochondrial proteome such as any one or more of the proteins having amino acid sequences set forth in SEQ ID NOS:1-3025) and identifying the cellular component(s) that binds to the compound to form a molecular complex, preferably through a specific interaction.

[0056] “Altered mitochondrial function” may refer to any condition or state, including those that accompany a disease associated with altered mitochondrial function, where any structure or activity that is directly or indirectly related to a mitochondrial function has been changed in a statistically significant manner relative to a control or standard. Altered mitochondrial function may have its origin in extramitochondrial structures or events as well as in mitochondrial structures or events, in direct interactions between mitochondrial and extramitochondrial genes and/or their gene products, or in structural or functional changes that occur as the result of interactions between intermediates that may be formed as the result of such interactions, including metabolites, catabolites, substrates, precursors, cofactors and the like.

[0057] Additionally, altered mitochondrial function may include altered respiratory, metabolic or other biochemical or biophysical activity in one or more cells of a biological sample or a biological source. As non-limiting examples, markedly impaired ETC activity may be related to altered mitochondrial function, as may be generation of increased reactive oxygen species (ROS) or defective oxidative phosphorylation. As further examples, altered mitochondrial membrane potential, induction of apoptotic pathways and formation of a typical chemical and biochemical crosslinked species within a cell, whether by enzymatic or non-enzymatic mechanisms, may all be regarded as indicative of altered mitochondrial function. These and other non-limiting examples of altered mitochondrial function are contemplated by the present invention.

[0058] For instance, altered mitochondrial function may be related, interalia, to altered intracellular calcium regulation that may accompany loss of mitochondrial membrane electrochemical potential by intracellular calcium flux, by mechanisms that include free radical oxidation, defects in transmitochondrial membrane shuttles and transporters such as the adenine nucleotide transporter or the malate-aspartate shuttle, by defects in ATP biosynthesis, by impaired association of hexokinases and/or other enzymes with porin at the inner mitochondrial membrane, or by other events. Altered intracellular calcium regulation and/or collapse of mitochondrial inner membrane potential may result from direct or indirect effects of mitochondrial genes, gene products or related downstream mediator molecules and/or extramitochondrial genes, gene products or related downstream mediators, or from other known or unknown causes.

[0059] Thus, an “indicator of altered mitochondrial function” may be any detectable parameter that directly relates to a condition, process, pathway, dynamic structure, state or other activity involving mitochondria and that permits detection of altered mitochondrial function in a biological sample from a subject or biological source. According to non-limiting theory, altered mitochondrial function therefore may also include altered mitochondrial permeability to calcium or to mitochondrial molecular components involved in apoptosis (e.g., cytochrome c), or other alterations in mitochondrial respiration, or any other altered biological activity as provided herein that is a mitochondrially associated activity.

[0060] In certain preferred embodiments of the invention, an enzyme is the indicator of altered mitochondrial function as provided herein. The enzyme may be a mitochondrial enzyme, which may further be an ETC enzyme or a Krebs cycle enzyme. The enzyme may also be an ATP biosynthesis factor, which may include an ETC enzyme and/or a Krebs cycle enzyme, or other enzymes or cellular components related to ATP production as provided herein. A “non-enzyme” refers to an indicator of altered mitochondrial function that is not an enzyme (i.e., that is not a mitochondrial enzyme or an ATP biosynthesis factor as provided herein). In certain other preferred embodiments, an enzyme is a co-indicator of altered mitochondrial function. The following enzymes may not be indicators of altered mitochondrial function according to the present invention, but may be co-indicators of altered mitochondrial function as provided herein: citrate synthase (EC 4.1.3.7), hexokinase II (EC 2.7.1.1; see, e.g., Kruszynska et al. 1.998), cytochrome c oxidase (EC 1.9.3.1), phosphofructokinase (EC 2.7.1.11), glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12), glycogen phosphorylase (EC 2.4.1.1) creatine kinase (EC 2.7.3.2), NADH dehydrogenase (EC 1.6.5.3), glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), triose phosphate dehydrogenase (EC 1.2.1.12) and malate dehydrogenase (EC 1.1.1.37).

[0061] In other highly preferred embodiments, the indicator of altered mitochondrial function is any ATP biosynthesis factor as described below. In other preferred embodiments, the indicator is ATP production. In other preferred embodiments, the indicator of altered mitochondrial function may be mitochondrial mass or mitochondrial number. According to the present invention, mitochondrial DNA content may not be an indicator of altered mitochondrial function but may be a co-predictor of altered mitochondrial function or a co-indicator of altered mitochondrial function, as provided herein. In other preferred embodiments the indicator of altered mitochondrial function may be free radical production, a cellular response to elevated intracellular calcium or a cellular response to an apoptogen.

[0062] INDICATORS OF ALTERED MITOCHONDRIAL FUNCTION THAT ARE ENZYMES

[0063] As provided herein, in certain preferred embodiments, an altered biological activity comprises an indicator of altered mitochondrial function that may be an enzyme; such an enzyme may be a mitochondrial enzyme or an ATP biosynthesis factor that is an enzyme, for example an ETC enzyme or a Krebs cycle enzyme.

[0064] Reference herein to “enzyme quantity”, “enzyme catalytic activity” or “enzyme expression level” is meant to include a reference to any of a mitochondrial enzyme quantity, activity or expression level or an ATP biosynthesis factor quantity, activity or expression level; either of which may further include, for example, an ETC enzyme quantity, activity or expression level or a Krebs cycle enzyme quantity, activity or expression level. In the most preferred embodiments of the invention, an enzyme is a natural or recombinant protein or polypeptide that has enzyme catalytic activity as provided herein. Such an enzyme may be, by way of non-limiting examples, an enzyme, a holoenzyme, an enzyme complex, an enzyme subunit, an enzyme fragment, derivative or analog or the like, including a truncated, processed or cleaved enzyme.

[0065] A “mitochondrial enzyme” that may be an indicator of altered mitochondrial function as provided herein refers to a mitochondrial molecular component that has enzyme catalytic activity and/or functions as an enzyme cofactor capable of influencing enzyme catalytic activity. As used herein, mitochondria are comprised of “mitochondrial molecular components”, which may be a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof, a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any covalently or non-covalently complexed combination of these components, or any other biological molecule that is a stable or transient constituent of a mitochondrion.

[0066] A mitochondrial enzyme that may be an indicator of altered mitochondrial function or a co-indicator of altered mitochondrial function as provided herein, or an ATP biosynthesis factor that may be an indicator of altered mitochondrial function as provided herein, may comprise an ETC enzyme, which refers to any mitochondrial molecular component that is a mitochondrial enzyme component of the mitochondrial electron transport chain (ETC) complex associated with the inner mitochondrial membrane and mitochondrial matrix. An ETC enzyme may include any of the multiple ETC subunit polypeptides encoded by mitochondrial and nuclear genes. The ETC is typically described as comprising complex I (NADH:ubiquinone reductase), complex II (succinate dehydrogenase), complex III (ubiquinone: cytochrome c oxidoreductase), complex IV (cytochrome c oxidase) and complex V (mitochondrial ATP synthetase), where each complex includes multiple polypeptides and cofactors (for review see, e.g., Walker et al., 1995 Meths. Enzymol. 260:14; Ernster et al., 1981 J. Cell Biol. 91:227s-255s, and references cited therein).

[0067] A mitochondrial enzyme that may be an indicator of altered mitochondrial function as provided herein, or an ATP biosynthesis factor that may be an indicator of altered mitochondrial function as provided herein, may also comprise a Krebs cycle enzyme, which includes mitochondrial molecular components that mediate the series of biochemical/bioenergetic reactions also known as the citric acid cycle or the tricarboxylic acid cycle (see, e.g., Lehninger, Biochemistry, 1975 Worth Publishers, NY; Voet and Voet, Biochemistry, 1990 John Wiley & Sons, NY; Mathews and van Holde, Biochemistry, 1990 Benjamin Cummings, Menlo Park, Calif.). Krebs cycle enzymes include subunits and cofactors of citrate synthase, aconitase, isocitrate dehydrogenase, the α-ketoglutarate dehydrogenase complex, succinyl CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase. Krebs cycle enzymes further include enzymes and cofactors that are functionally linked to the reactions of the Krebs cycle, such as, for example, nicotinamide adenine dinucleotide, coenzyme A, thiamine pyrophosphate, lipoamide, guanosine diphosphate, flavin adenine dinucloetide, acetyl-coA carboxylase (ACC) and nucleoside diphosphokinase.

[0068] The methods of the present invention also pertain in part to the correlation of mitochondrial associated disease with an indicator of altered mitochondrial function that may be an ATP biosynthesis factor, an altered amount of ATP or an altered amount of ATP production.

[0069] An “ATP biosynthesis factor” refers to any naturally occurring cellular component that contributes to the efficiency of ATP production in mitochondria. Such a cellular component may be a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof, a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like. An ATP biosynthesis factor includes at least the components of the ETC and of the Krebs cycle (see, e.g., Lehninger, Biochemistry, 1975 Worth Publishers, NY; Voet and Voet, Biochemistry, 1990 John Wiley & Sons, NY; Mathews and van Holde, Biochemistry, 1990 Benjamin Cummings, Menlo Park, Calif.) and any protein, enzyme or other cellular component that participates in ATP synthesis, regardless of whether such ATP biosynthesis factor is the product of a nuclear gene or of an extranuclear gene (e.g., a mitochondrial gene). Participation in ATP synthesis may include, but need not be limited to, catalysis of any reaction related to ATP synthesis, transmembrane import and/or export of ATP or of an enzyme cofactor, transcription of a gene encoding a mitochondrial enzyme and/or translation of such a gene transcript.

[0070] Compositions and methods for determining whether a cellular component is an ATP biosynthesis factor are well known in the art, and include methods for determining ATP production (including determination of the rate of ATP production in a sample) and methods for quantifying ATP itself. The contribution of an ATP biosynthesis factor to ATP production can be determined, for example, using an isolated ATP biosynthesis factor that is added to cells or to a cell-free system. The ATP biosynthesis factor may directly or indirectly mediate a step or steps in a biosynthetic pathway that influences ATP production. For example, an ATP biosynthesis factor may be an enzyme that catalyzes a particular chemical reaction leading to ATP production. As another example, an ATP biosynthesis factor may be a cofactor that enhances the efficiency of such an enzyme. As another example, an ATP biosynthesis factor may be an exogenous genetic element introduced into a cell or a cell-free system that directly or indirectly affects an ATP biosynthetic pathway. Those having ordinary skill in the art are readily able to compare ATP production by an ATP biosynthetic pathway in the presence and absence of a candidate ATP biosynthesis factor. Routine determination of ATP production may be accomplished using any known method for quantitative ATP detection, for example by way of illustration and not limitation, by differential extraction from a sample optionally including chromatographic isolation; by spectrophotometry; by quantification of labeled ATP recovered from a sample contacted with a suitable form of a detectably labeled ATP precursor molecule such as, for example, 32P; by quantification of an enzyme activity associated with ATP synthesis or degradation; or by other techniques that are known in the art. Accordingly, in certain embodiments of the present invention, the amount of ATP in a biological sample or the production of ATP (including the rate of ATP production) in a biological sample may be an indicator of altered mitochondrial function. In one embodiment, for instance, ATP may be quantified by measuring luminescence of luciferase catalyzed oxidation of D-luciferin, an ATP dependent process.

[0071] “Enzyme catalytic activity” refers to any function performed by a particular enzyme or category of enzymes that is directed to one or more particular cellular function(s). For example, “ATP biosynthesis factor catalytic activity” refers to any function performed by an ATP biosynthesis factor as provided herein that contributes to the production of ATP. Typically, enzyme catalytic activity is manifested as facilitation of a chemical reaction by a particular enzyme, for instance an enzyme that is an ATP biosynthesis factor, wherein at least one enzyme substrate or reactant is covalently modified to form a product. For example, enzyme catalytic activity may result in a substrate or reactant being modified by formation or cleavage of a covalent chemical bond, but the invention need not be so limited. Various methods of measuring enzyme catalytic activity are known to those having ordinary skill in the art and depend on the particular activity to be determined.

[0072] For many enzymes, including mitochondrial enzymes or enzymes that are ATP biosynthesis factors as provided herein, quantitative criteria for enzyme catalytic activity are well established. These criteria include, for example, activity that may be defined by international units (IU), by enzyme turnover number, by catalytic rate constant (Kcat), by Michaelis-Menten constant (Km), by specific activity or by any other enzymological method known in the art for measuring a level of at least one enzyme catalytic activity. Specific activity of a mitochondrial enzyme, such as an ATP biosynthesis factor, may be expressed as units of substrate detectably converted to product per unit time and, optionally, further per unit sample mass (e.g., per unit protein or per unit mitochondrial mass).

[0073] In certain preferred embodiments of the invention, enzyme catalytic activity may be expressed as units of substrate detectably converted by an enzyme to a product per unit time per unit total protein in a sample. In certain particularly preferred embodiments, enzyme catalytic activity may be expressed as units of substrate detectably converted by an enzyme to product per unit time per unit mitochondrial mass in a sample. In certain highly preferred embodiments, enzyme catalytic activity may be expressed as units of substrate detectably converted by an enzyme to product per unit time per unit mitochondrial protein mass in a sample. Products of enzyme catalytic activity may be detected by suitable methods that will depend on the quantity and physicochemical properties of the particular product. Thus, detection may be, for example by way of illustration and not limitation, by radiometric, calorimetric, spectrophotometric, fluorimetric, immunometric or mass spectrometric procedures, or by other suitable means that will be readily apparent to a person having ordinary skill in the art.

[0074] In certain embodiments of the invention, detection of a product of enzyme catalytic activity may be accomplished directly, and in certain other embodiments detection of a product may be accomplished by introduction of a detectable reporter moiety or label into a substrate or reactant such as a marker enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, or the like. The amount of such a label that is present as unreacted substrate and/or as reaction product, following a reaction to assay enzyme catalytic activity, is then determined using a method appropriate for the specific detectable reporter moiety or label. For radioactive groups, radionuclide decay monitoring, scintillation counting, scintillation proximity assays (SPA) or autoradiographic methods are generally appropriate. For immunometric measurements, suitably labeled antibodies may be prepared including, for example, those labeled with radionuclides, with fluorophores, with affinity tags, with biotin or biotin mimetic sequences or those prepared as antibody-enzyme conjugates (see, e.g., Weir, D. M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; Scouten, W. H., Methods in Enzymology 135:30-65,1987; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals-Sixth Ed., Molecular Probes, Eugene, Oreg.; Scopes, R. K., Protein Purification: Principles and Practice, 1987, Springer-Verlag, NY; Hermanson, G. T. et al., Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., NY; Luo et al., 1998 J. Biotechnol. 65:225 and references cited therein). Spectroscopic methods may be used to detect dyes (including, for example, calorimetric products of enzyme reactions), luminescent groups and fluorescent groups. Biotin may be detected using avidin or streptavidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic, spectrophotometric or other analysis of the reaction products. Standards and standard additions may be used to determine the level of enzyme catalytic activity in a sample, using well known techniques.

[0075] As noted above, enzyme catalytic activity of an ATP biosynthesis factor may further include other functional activities that lead to ATP production, beyond those involving covalent alteration of a substrate or reactant. For example by way of illustration and not limitation, an ATP biosynthesis factor that is an enzyme may refer to a transmembrane transporter molecule that, through its enzyme catalytic activity, facilitates the movement of metabolites between cellular compartments. Such metabolites may be-ATP or other cellular components involved in ATP synthesis, such as gene products and their downstream intermediates, including metabolites, catabolites, substrates, precursors, cofactors and the like. As another non-limiting example, an ATP biosynthesis factor that is an enzyme may, through its enzyme catalytic activity, transiently bind to a cellular component involved in ATP synthesis in a manner that promotes ATP synthesis. Such a binding event may, for instance, deliver the cellular component to another enzyme involved in ATP synthesis and/or may alter the conformation of the cellular component in a manner that promotes ATP synthesis. Further to this example, such conformational alteration may be part of a signal transduction pathway, an allosteric activation pathway, a transcriptional activation pathway or the like, where an interaction between cellular components leads to ATP production.

[0076] Thus, according to the present invention, an ATP biosynthesis factor may include, as non-limiting examples, an ATP synthase, acetyl-coA carboxylase (ACC) a mitochondrial matrix protein and a mitochondrial membrane protein. Suitable mitochondrial membrane proteins include such mitochondrial components as the adenine nucleotide transporter (ANT; e.g., Fiore et al., 1998 Biochimie 80:137; Klingenberg 1985 Ann. N.Y. Acad. Sci. 456:279), the voltage dependent anion channel (VDAC, also referred to as porin; e.g., Manella, 1997 J. Bioenergetics Biomembr. 29:525), the malate-aspartate shuttle, the mitochondrial calcium uniporter (e.g., Litsky et al., 1997 Biochem. 36:7071), uncoupling proteins (UCP-1, -2, -3; see e.g., Jezek et al., 1998 Int. J. Biochem. Cell Biol. 30:1163), a hexokinase, a peripheral benzodiazepine receptor, a mitochondrial intermembrane creatine kinase, cyclophilin D, a Bcl-2 gene family encoded polypeptide, the tricarboxylate carrier (e.g., lacobazi et al., 1996 Biochim. Biophys. Acta 1284:9; Bisaccia et al., 1990 Biochim. Biophys. Acta 1019:250) and the dicarboxylate carrier (e.g., Fiermonte et al., 1998 J. Biol. Chem. 273:24754; Indiveri et al., 1993 Biochim. Biophys. Acta 1143:310; for a general review of mitochondrial membrane transporters, see, e.g., Zoratti et al., 1994 J. Bioenergetics Biomembr. 26:543 and references cited therein).

[0077] “Enzyme quantity” as used herein refers to an amount of an enzyme including mitochondrial enzymes or enzymes that are ATP biosynthesis factors as provided herein, or of another ATP biosynthesis factor, that is present, i.e., the physical presence of an enzyme or ATP biosynthesis factor selected as an indicator of altered mitochondrial function, irrespective of enzyme catalytic activity. Depending on the physicochemical properties of a particular enzyme or ATP biosynthesis factor, the preferred method for determining the enzyme quantity will vary. In the most highly preferred embodiments of the invention, determination of enzyme quantity will involve quantitative determination of the level of a protein or polypeptide using routine methods in protein chemistry with which those having skill in the art will be readily familiar, for example by way of illustration and not limitation, those described in greater detail below.

[0078] Accordingly, determination of enzyme quantity may be by any suitable method known in the art for quantifying a particular cellular component that is an enzyme or an ATP biosynthesis factor as provided herein, and that in preferred embodiments is a protein or polypeptide. Depending on the nature and physicochemical properties of the enzyme or ATP biosynthesis factor, determination of enzyme quantity may be by densitometric, mass spectrometric, spectrophotometric, fluorimetric, immunometric, chromatographic, electrochemical or any other means of quantitatively detecting a particular cellular component. Methods for determining enzyme quantity also include methods described above that are useful for detecting products of enzyme catalytic activity, including those measuring enzyme quantity directly and those measuring a detectable label or reporter moiety. In certain preferred embodiments of the invention, enzyme quantity is determined by immunometric measurement of an isolated enzyme or ATP biosynthesis factor. In certain preferred embodiments of the invention, these and other immunological and immunochemical techniques for quantitative determination of biomolecules such as an enzyme or ATP biosynthesis factor may be employed using a variety of assay formats known to those of ordinary skill in the art, including but not limited to enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion and other techniques. (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Weir, D. M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston.) For example, the assay may be performed in a Western blot format, wherein a preparation comprising proteins from a biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react with an antibody specific for an enzyme or an ATP biosynthesis factor that is a protein or polypeptide. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as is well known in the art and described above.

[0079] INDICATORS OF ALTERED MITOCHONDRIAL FUNCTION THAT ARE CELLULAR RESPONSES TO ELEVATED INTRACELLULAR CALCIUM

[0080] According to certain embodiments of the present invention, a method is provided that comprises in pertinent part determining a biological activity of a mitochondrial polypeptide by monitoring intracellular calcium homeostasis and/or cellular responses to perturbations of this homeostasis, including physiological and pathophysiological calcium regulation. In particular, according to these embodiments, the method of the present invention is directed to comparing a cellular response to elevated intracellular calcium in a biological sample in the presence and absence of a candidate agent, or to comparing such a response in a sample from a subject known or suspected of having a disease associated with altered mitochondrial function with that of a control subject. The range of cellular responses to elevated intracellular calcium is broad, as is the range of methods and reagents for the detection of such responses. Many specific cellular responses are known to those having ordinary skill in the art; these responses will depend on the particular cell types present in a selected biological sample. It is within the contemplation of the present invention to provide a method comprising comparing a cellular response to elevated intracellular calcium, where such response is an indicator of altered mitochondrial function as provided herein. As non-limiting examples, cellular responses to elevated intracellular calcium include secretion of specific secretory products, exocytosis of particular preformed components, increased glycogen metabolism and cell proliferation (see, e.g., Clapham, 1995 Cell 80:259; Cooper, The Cell—A Molecular Approach, 1997 ASM Press, Washington, D.C.; Alberts, B., Bray, D., et al., Molecular Biology of the Cell, 1995 Garland Publishing, NY).

[0081] As a brief background, normal alterations of intramitochondrial Ca2+ are associated with normal metabolic regulation (Dykens, 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp.29-55; Radi et al., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 57-89; Gunter and Pfeiffer, 1991, Am. J. Physiol. 27: C755; Gunter et al., 1994, Am. J. Physiol. 267: 313). For example, fluctuating levels of mitochondrial free Ca2+ may be responsible for regulating oxidative metabolism in response to increased ATP utilization, via allosteric regulation of enzymes (reviewed by Crompton et al., 1993 Basic Res. Cardiol. 88: 513-523;) and the glycerophosphate shuttle (Gunter et al., 1994 J. Bioenerg. Biomembr. 26: 471).

[0082] Normal mitochondrial function includes regulation of cytosolic free calcium levels by sequestration of excess Ca2+ within the mitochondrial matrix. Depending on cell type, cytosolic Ca2+ concentration is typically 50-100 nM. In normally functioning cells, when Ca2+ levels reach 200-300 nM, mitochondria begin to accumulate Ca2+ as a function of the equilibrium between influx via a Ca2+ uniporter in the inner mitochondrial membrane and Ca2+ efflux via both Na+ dependent and Na+ independent calcium carriers. In certain instances, such perturbation of intracellular calcium homeostasis is a feature of diseases associated with altered mitochondrial function, regardless of whether the calcium regulatory dysfunction is causative of, or a consequence of, altered mitochondrial function.

[0083] Elevated mitochondrial calcium levels thus may accumulate in response to an initial elevation in cytosolic free calcium, as described above. Such elevated mitochondrial calcium concentrations in combination with reduced ATP or other conditions associated with mitochondrial pathology, can lead to collapse of mitochondrial inner membrane potential (see Gunter et al., 1998 Biochim. Biophys. Acta 1366:5; Rottenberg and Marbach, 1990, Biochim. Biophys. Acta 1016:87). Generally, in order to practice the subject invention methods, the extramitochondrial (cytosolic) level of Ca2+ in a biological sample is greater than that present within mitochondria. For example, in the case of type 2 diabetes mellitus (type 2 DM), mitochondrial or cytosolic calcium levels may vary from the above ranges and may range from, e.g., about 1 nM to about 500 mM, more typically from about 10 nM to about 100 μM and usually from about 20 nM to about 1 μM, where “about” indicates ±10%. A variety of calcium indicators are known in the art, including but not limited to, for example, fura-2 (McCormack et al., 1989 Biochim. Biophys. Acta 973:420); magfura-2; BTC (U.S. Pat. No. 5,501,980); fluo-3, fluo-4 and fluo-5N (U.S. Pat. No. 5,049,673); rhod-2; benzothiaza-1; and benzothiaza-2 (all of which are available from Molecular Probes, Eugene, Oreg.). These or any other means for monitoring intracellular calcium are contemplated according to the subject invention method for identifying a risk for type 2 DM.

[0084] For monitoring an indicator of altered mitochondrial function that is a cellular response to elevated intracellular calcium, compounds that induce increased cytoplasmic and mitochondrial concentrations of Ca2+, including calcium ionophores, are well known to those of ordinary skill in the art, as are methods for measuring intracellular calcium and intramitochondrial calcium (see, e.g., Gunter and Gunter, 1994 J. Bioenerg. Biomembr. 26: 471; Gunter et al., 1998 Biochim. Biophys. Acta 1366:5; McCormack et al., 1989 Biochim. Biophys. Acta 973:420; Orrenius and Nicotera, 1994 J. Neural. Transm. Suppl. 43:1; Leist and Nicotera, 1998 Rev. Physiol. Biochem. Pharmacol. 132:79; and Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals-Sixth Ed., Molecular Probes, Eugene, Oreg.). Accordingly, a person skilled in the art may readily select a suitable ionophore (or another compound that results in increased cytoplasmic and/or mitochondrial concentrations of Ca2+) and an appropriate means for detecting intracellular and/or intramitochondrial calcium for use in the present invention, according to the instant disclosure and to well known methods.

[0085] Ca2+ influx into mitochondria appears to be largely dependent, and may be completely dependent, upon the negative transmembrane electrochemical potential (Δψ) established at the inner mitochondrial membrane by electron transfer, and such influx fails to occur in the absence of Δψ even when an eight-fold Ca2+ concentration gradient is imposed (Kapus et al., 1991 FEBS Lett. 282:61). Accordingly, mitochondria may release Ca2+ when the membrane potential is dissipated, as occurs with uncouplers like 2,4-dinitrophenol and carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP). Thus, according to certain embodiments of the present invention, collapse of Δψ may be potentiated by influxes of cytosolic free calcium into the mitochondria, as may occur under certain physiological conditions including those encountered by cells of a subject having type 2 DM. Detection of such collapse may be accomplished by a variety of means as provided herein.

[0086] Typically, mitochondrial membrane potential may be determined according to methods with which those skilled in the art will be readily familiar, including but not limited to detection and/or measurement of detectable compounds such as fluorescent indicators, optical probes and/or sensitive pH and ion-selective electrodes (See, e.g., Ernster et al., 1981 J. Cell Biol. 91:227s and references cited; see also Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals-Sixth Ed., Molecular Probes, Eugene, Oreg., pp.266-274 and 589-594.). For example, by way of illustration and not limitation, the fluorescent probes 2-,4-dimethylaminostyryl-N-methyl pyridinium (DASPMI) and tetramethylrhodamine esters (such as, e.g., tetramethylrhodamine methyl ester, TMRM; tetramethylrhodamine ethyl ester, TMRE) or related compounds (see, e.g., Haugland, 1996, supra) may be quantified following accumulation in mitochondria, a process that is dependent on, and proportional to, mitochondrial membrane potential (see, e.g., Murphy et al., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp.159-186 and references cited therein; and Molecular Probes On-line Handbook of Fluorescent Probes and Research Chemicals, at http://www.probes.com/handbook/toc.html). Other fluorescent detectable compounds that may be used in the invention include but are not limited to rhodamine 123, rhodamine B hexyl ester, DiOC6(3), JC-1 [5,5′,6,6′-Tetrachloro-1,1′,3,3′-Tetraethylbezimidazolcarbocyanine Iodide] (see Cossarizza, et al., 1993 Biochem. Biophys. Res. Comm. 197:40; Reers et al., 1995 Meth. Enzymol. 260:406), rhod-2 (see U.S. Pat. No. 5,049,673; all of the preceding compounds are available from Molecular Probes, Eugene, Oreg.) and rhodamine 800 (Lambda Physik, GmbH, Göttingen, Germany; see Sakanoue et al., 1997 J. Biochem. 121:29). Methods for monitoring mitochondrial membrane potential are also disclosed in U.S. application Ser. No. 09/161,172.

[0087] Mitochondrial membrane potential can also be measured by nonfluorescent means, for example by using TTP (tetraphenylphosphonium ion) and a TTP-sensitive electrode (Kamo et al., 1979 J. Membrane Biol. 49:105; Porter and Brand, 1995 Am. J. Physiol. 269:R1213). Those skilled in the art will be able to select appropriate detectable compounds or other appropriate means for measuring Δψm. By way of example and not limitation, TMRM is somewhat preferable to TMRE because, following efflux from mitochondria, TMRE yields slightly more residual signal in the endoplasmic reticulicum and cytoplasm than TMRM.

[0088] As another non-limiting example, membrane potential may be additionally or alternatively calculated from indirect measurements of mitochondrial permeability to detectable charged solutes, using matrix volume and/or pyridine nucleotide redox determination combined with spectrophotometric or fluorimetric quantification. Measurement of membrane potential dependent substrate exchange-diffusion across the inner mitochondrial membrane may also provide an indirect measurement of membrane potential. (See, e.g., Quinn, 1976, The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Md., pp. 200-217 and references cited therein.)

[0089] Exquisite sensitivity to extraordinary mitochondrial accumulations of Ca2+ that result from elevation of intracellular calcium, as described above, may also characterize type 2 DM. Such mitochondrial sensitivity may provide an indicator of altered mitochondrial function according to the present invention. Additionally, a variety of physiologically pertinent agents, including hydroperoxide and free radicals, may synergize with Ca2+ to induce collapse of Δψ (Novgorodov et al., 1991 Biochem. Biophys. Acta 1058: 242; Takeyama et al., 1993 Biochem. J. 294:719; Guidox et al., 1993 Arch. Biochem. Biophys. 306:139).

[0090] INDICATORS OF ALTERED MITOCHONDRIAL FUNCTION THAT ARE CELLULAR RESPONSES TO APOPTOGENIC STIMULI

[0091] Turning to another aspect, the present invention relates to the correlation of diseases associated with altered mitochondrial function with an indicator of altered mitochondrial function, involving programmed cell death or apoptosis. In particular, according to this aspect, the present invention is directed to a method comprising comparing a cellular response to an apoptosis-inducing (“apoptogenic”) stimulus in a biological sample from (i) a subject believed to be at risk for disease, and (ii) a control subject. The range of cellular responses to various known apoptogenic stimuli is broad, as is the range of methods and reagents for the detection of such responses. It is within the contemplation of the present invention to provide a method for identifying a risk for disease by comparing a cellular response to an apoptogenic stimulus, where such response is an indicator of altered mitochondrial function as provided herein.

[0092] By way of background, mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995). Altered mitochondrial physiology may be among the earliest events in programmed cell death (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42,1994). In several cell types, reduction in the mitochondrial membrane potential (Δψm) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratory activity leading to altered cellular metabolic states, such as elevated intracellular ROS, may occur in certain diseases associated with altered mitochondrial function (e.g., type 2 DM) and may further induce pathogenetic events via apoptotic mechanisms.

[0093] Oxidatively stressed mitochondria may release a pre-formed soluble factor that can induce chromosomal condensation, an event preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). In addition, members of the Bcl-2 family of anti-apoptosis gene products are located within the outer mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem. 40:1819-25,1992) and these proteins appear to protect membranes from oxidative stress (Korsmeyer et al, Biochim. Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membrane appears to be indispensable for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrial physiology may be important mediators of apoptosis.

[0094] Altered mitochondrial function, may therefore lower the threshold for induction of apoptosis by an apoptogen. A variety of apoptogens are known to those familiar with the art (see, e.g., Green et al., 1998 Science 281:1309 and references cited therein) and may include by way of illustration and not limitation: tumor necrosis factor-alpha (TNF-α); Fas ligand; glutamate; N-methyl-D-aspartate (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al., 1997 J. Cell. Biol. 138:449-469); paraquat (Costantini et al., 1995 Toxicology 99:1-2); ethylene glycols; protein kinase inhibitors, such as, e.g. staurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrine chloride, genistein; 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; N-[2-((p-bromocinnamyl)amino)ethyl]-5-5-isoquinolinesulfonamide; KN-93; quercitin; d-erythro-sphingosine derivatives; UV irradiation; ionophores such as, e.g.: ionomycin and valinomycin; MAP kinase inducers such as, e.g.: anisomycin, anandamine; cell cycle blockers such as, e.g.: aphidicolin, colcemid, 5-fluorouracil, homoharringtonine; acetylcholinesterase inhibitors such as, e.g. berberine; anti-estrogens such as, e.g.: tamoxifen; pro-oxidants, such as, e.g.,: tert-butyl peroxide, hydrogen peroxide; free radicals such as, e.g., nitric oxide; inorganic metal ions, such as, e.g., cadmium; DNA synthesis inhibitors such as, e.g.: actinomycin D; DNA intercalators such as, e.g., doxorubicin, bleomycin sulfate, hydroxyurea, methotrexate, mitomycin C, camptothecin, daunorubicin; protein synthesis inhibitors such as, e.g., cycloheximide, puromycin, rapamycin; agents that affect microtubulin formation or stability such as, e.g.: vinblastine, vincristine, colchicine, 4-hydroxyphenylretinamide, paclitaxel; Bad protein, Bid protein and Bax protein (see, e.g., Jurgenmeier et al., 1998 Proc. Nat. Acad. Sci. USA 95:4997-5002 and references cited therein); calcium and inorganic phosphate (Kroemer et al., 1998 Ann. Rev. Physiol. 60:619).

[0095] In one embodiment of the subject invention method wherein the indicator of altered mitochondrial function is a cellular response to an apoptogen, cells in a biological sample that are suspected of undergoing apoptosis may be examined for morphological, permeability or other changes that are indicative of an apoptotic state. For example by way of illustration and not limitation, apoptosis in many cell types may cause altered morphological appearance such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by a person having ordinary skill in the art, for example by using light microscopy. As another example, cells undergoing apoptosis may exhibit fragmentation and disintegration of chromosomes, which may be apparent by microscopy and/or through the use of DNA-specific or chromatin-specific dyes that are known in the art, including fluorescent dyes. Such cells may also exhibit altered plasma membrane permeability properties as may be readily detected through the use of vital dyes (e.g., propidium iodide, trypan blue) or by the detection of lactate dehydrogenase leakage into the extracellular milieu. These and other means for detecting apoptotic cells by morphologic criteria, altered plasma membrane permeability and related changes will be apparent to those familiar with the art.

[0096] In another embodiment of the subject invention method wherein the indicator of altered mitochondrial function is a cellular response to an apoptogen, cells in a biological sample may be assayed for translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane, which may be detected, for example, by measuring outer leaflet binding by the PS-specific protein annexin. (Martin et al., J. Exp. Med. 182:1545,1995; Fadok et al., J. Immunol. 148:2207,1992.) In still another embodiment of this aspect of the invention, a cellular response to an apoptogen is determined by an assay for induction of specific protease activity in any member of a family of apoptosis-activated proteases known as the caspases (see, e.g., Green et al., 1998 Science 281:1309). Those having ordinary skill in the art will be readily familiar with methods for determining caspase activity, for example by determination of caspase-mediated cleavage of specifically recognized protein substrates. These substrates may include, for example, poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that are known in the art (see, e.g., Ellerby et al., 1997 J. Neurosci. 17:6165). The synthetic peptide Z-Tyr-Val-Ala-Asp-AFC (SEQ ID NO: ______;), wherein “Z” indicates a benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science 275:1132; Nicholson et al., 1995 Nature 376:37), is one such substrate. Other non-limiting examples of substrates include nuclear proteins such as U1-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997 J. Cell. Biochem. 64:50; Cohen, 1997 Biochem. J. 326:1).

[0097] As described above, the mitochondrial inner membrane may exhibit highly selective and regulated permeability for many small solutes, but is impermeable to large (>˜10 kDa) molecules. (See, e.g., Quinn, 1976 The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Md.). In cells undergoing apoptosis, however, collapse of mitochondrial membrane potential may be accompanied by increased permeability permitting macromolecule diffusion across the mitochondrial membrane. Thus, in another embodiment of the subject invention method wherein the indicator of altered mitochondrial function is a cellular response to an apoptogen, detection of a mitochondrial protein, for example cytochrome c that has escaped from mitochondria in apoptotic cells, may provide evidence of a response to an apoptogen that can be readily determined. (Liu et al., Cell 86:147, 1996) Such detection of cytochrome c may be performed spectrophotometrically, immunochemically or by other well established methods for determining the presence of a specific protein.

[0098] For instance, release of cytochrome c from cells challenged with apoptotic stimuli (e.g., ionomycin, a well known calcium ionophore) can be followed by a variety of immunological methods. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry coupled with affinity capture is particularly suitable for such analysis since apo-cytochrome c and holo-cytochrome c can be distinguished on the basis of their unique molecular weights. For example, the Surface-Enhanced Laser Desorption/lonization (SELDI™) system (Ciphergen, Palo Alto, Calif.) may be utilized to detect cytochrome c release from mitochondria in apoptogen treated cells. In this approach, a cytochrome c specific antibody immobilized on a solid support is used to capture released cytochrome c present in a soluble cell extract. The captured protein is then encased in a matrix of an energy absorption molecule (EAM) and is desorbed from the solid support surface using pulsed laser excitation. The molecular mass of the protein is determined by its time of flight to the detector of the SELDI™ mass spectrometer.

[0099] A person having ordinary skill in the art will readily appreciate that there may be other suitable techniques for quantifying apoptosis, and such techniques for purposes of determining an indicator of altered mitochondrial function that is a cellular response to an apoptogenic stimulus are within the scope of the methods provided by the present invention.

[0100] As noted above, an increasing number of diseases, disorders and conditions have been identified as diseases associated with altered mitochondrial function as provided herein, such that given the present disclosure and the state of the art with respect to methods for assessing mitochondrial function and with respect to clinical signs and symptoms of such diseases, the person having ordinary skill in the art can readily determine criteria for establishing a statistically significant deviation from a normal range for one or more parameters that are appropriate to the definition of the disease, in order to establish that a disease associated with altered mitochondrial function is present. As an illustrative example, where it is desirable to determine whether or not a subject or biological source falls within clinical parameters indicative of type 2 diabetes mellitus, signs and symptoms of type 2 diabetes that are accepted by those skilled in the art may be used to so designate a subject or biological source, for example clinical signs referred to in Gavin et al. (Diabetes Care 22(suppl. 1):S5-S19,1999, American Diabetes Association Expert Committee on the Diagnosis and Classification of Diabetes Mellitus) and references cited therein, or other means known in the art for diagnosing type 2 diabetes. Similarly, those familiar with the art will be aware of art accepted criteria for determining the presence of other diseases associated with altered mitochondrial function as provided herein.

[0101] Hence, the person having ordinary skill in the art can “correlate” one or more parameters described herein (e.g., mitochondrial functions) with such a disease associated with altered mitochondrial function, in view of the present disclosure and based on familiarity with the art. Briefly, statistically significant deviation from a normal, disease-free range for any of a number of clinical signs and symptoms and/or criteria for mitochondrial function, permits determination of the statistically significant coincidence of such parameter(s) with disease. Such deviation can further be confirmed, for instance, by comparing the same parameters and criteria that are detected in disease to those in a suitable control sample, in this case a control derived from a subject known to be free of a risk for having, or presence of, such disease.

[0102] Accordingly, given the disclosure of the instant application, and in particular the identification of the polypeptide sequences set forth in SEQ ID NOS:1-3025 as belonging to a defined human mitochondrial proteome, the present invention provides a control set of polypeptides such that a sample may be analyzed for the presence of at least one modified polypeptide as described herein, in order to so “correlate” such modification with a disease associated with altered mitochondrial function. Establishing such a correlation then provides a target for screening assays to identify an agent suitable for therapeutic intervention, i.e., an agent that beneficially counteracts the disease-associated alteration in mitochondrial function. Without wishing to be bound by theory, a target for therapeutic intervention preferably contributes to the pathogenesis of disease by exhibiting undesirably altered biological activity, such that a therapeutic agent reverses such alteration to a control range. The invention need not, however, be so limited, as even in situations where the target identified according to the subject invention method is a surrogate marker of disease, such a target nevertheless may be restored to a normal control range by a therapeutic agent regardless of whether the interaction is direct, in a manner that ameliorates disease. In certain embodiments the invention further provides for determination of altered biological activity in such a modified polypeptide, as also described herein.

[0103] According to the present invention, there are provided compositions and methods for the identification of differential protein expression at the organellar proteome level (e.g., the mitochondrial proteome), in a sub-proteomic, complex mixture of proteins or at the level of a single targeted protein. The invention thus relates in pertinent part to the unexpected advantages associated with the unique physicochemical properties of particular organelle-derived (e.g., mitochondria) polypeptides, peptides (e.g., peptide fragments) and proteins, in conjunction with biochemical (including immunochemical) methods, modern spectrometry and protein bioinformatics software tools to identify peptides and proteins that are detected as differentially expressed products, and to identify previously unrecognized peptides and proteins as molecular components of a particular organelle (e.g., mitochondrial molecular components as provided herein).

[0104] The invention also relates in pertinent part to the surprising advantages offered by the use of an organelle enriched sample fraction (e.g., a mitochondria enriched sample as provided herein). Determining the pattern of differential protein expression (e.g., absence or presence of one or more particular proteins in a sample; structural modification of a particular protein; or other altered expression such as a statistically significant increase or decrease in the amount of one or more particular proteins in a sample when normalized to a control) at the peptide and/or protein level in a complex protein mixture obtained from a biological sample as provided herein (i.e., at the proteomic level) provides, in certain embodiments, targets for drug screening assays and for therapeutic intervention in specific disease states. Accordingly, in certain embodiments the invention provides methods for evaluating the effects of candidate therapeutic agents (e.g., drugs or biological stimuli as provided herein) on biological activity of a mitochondrial protein, for example, where the protein exhibits altered biological activity due to one or more of a modification such as a mutation (insertion, deletion and/or substitution of one or more amino acids), a posttranslational modification or an altered level of protein expression. Thus, in certain embodiments, such candidate agents may cause one or more specific alterations (e.g., increases or decreases in a statistically significant manner) in the biological activity of a mitochondrial protein, preferably in some beneficial fashion.

[0105] As also noted elsewhere herein, certain embodiments of the invention relate in pertinent part to isolating at least one mitochondrial polypeptide according to any of a variety of biochemical separation methodologies for isolating a polypeptide as known in the art and as provided herein (see, e.g., Scopes, 1987 Protein Purification: Principles and Practice, Springer-Verlag, NY; Deutscher, 1990 Meths. Enzymol. Vol. 182; Nilsson et al., 2000 Mass Spectrom. Rev. 19:390; Godovac-Zimmermann et al., 2001 Mass Spectrom. Rev. 20:1; Gatlin et al., 2000 Anal. Chem. 72:757; Link et al., 1999 Nat. Biotechnol. 17:676). Hence, as provided herein and as known to the art, such methodologies for isolating a mitochondrial polypeptide may exploit physicochemical and hydrodynamic properties of the polypeptide, including, for example, the approximate apparent molecular mass of the polypeptide, the amino acid sequence of the polypeptide, and in certain contemplated embodiments, the apparent approximate isolelectric focusing point of the polypeptide.

[0106] As is well known to those having ordinary skill in the art, variability in biological sample source and condition, extraction reagents and methods, separation media and instrumentation, analytical apparatus and the like, may account for differences in values observed for such properties of polypeptides as molecular mass and isoelectric focusing point. Hence, it will be understood that an “apparent” molecular mass or isoelectric focusing point refers to that which is detected in a particular rendition of a particular isolation procedure, although the value detected for such a parameter may vary among separate isolations; similarly those familiar with the art will appreciate that from among the variables listed above, including imprecision in instrumentation, apparent values may vary in a manner that renders a particular value that is detected only an “approximation” of the actual parameter being measured. Thus, according to certain embodiments of the present invention a mitochondrial polypeptide may be isolated on the basis of approximate apparent molecular mass, apparent approximate isoelectric focusing point and/or amino acid sequence, which parameters may be susceptible to some variability for reasons discussed above but which, in any event, will permit isolation of such a polypeptide as provided herein.

[0107] The isolated polypeptide is then contacted with a proteolytic agent to generate a plurality of derivative peptide fragments, from which a mass spectrum can be generated to permit determination of the presence, amount or structure (e.g., level) of the polypeptide in the sample, which may then be compared to similarly obtained levels of a mitochondrial polypeptide obtained from other samples.

[0108] In an effort to better understand the molecular details of mitochondrial dysfunction as a contributing factor in disease, a high-resolution map of the human mitochondrial proteome is disclosed herein using human heart tissue as the source of isolated mitochondria, which are further enriched on metrizamide density gradients, solubilized and fractionated using sucrose density gradients. Although a protein map was previously generated using an only partially enriched mitochondrial fraction from human placenta (Rabilloud et al., 1998 Electrophor. 19:1006), no reliable database cataloguing mitochondrial proteins is currently available (cf., e.g., Koc et al., 2000 J. Biol. Chem. 275:32585; Lopez et al., 2000 Electrophor. 21:3427). Typically, mitochondria may be obtained from brain, heart, skeletal muscle or liver, where they are most abundant, although other sources (e.g., blood platelets) may also be used. According to the present invention there is provided a framework for investigating mitochondrial proteins, including identifying previously unrecognized mitochondrial proteins (e.g., novel proteins or known proteins not previously known to exist as mitochondrial molecular components) as well as those that are modified as provided herein as a correlate of disease, by mapping the human heart mitochondrial proteome. As described in greater detail in the Examples, mitochondrial proteins in distinct sucrose density gradient fractions were separated by one-dimensional polyacrylamide gel electrophoresis, and isolated proteins recovered from gels were analyzed as described below using matrix assisted laser desorption ionization (MALDI) and MALDI-post source decay (MALDI-PSD) techniques. (For other MS methods for proteins, see, e.g., Godovac-Zimmermann et al., 2001 Mass Spectromet. Rev. 20:1-57; Nilsson et al., 2000 Mass Spectromet. Rev. 19:390-397.) Over 1400 proteins were identified in the NCBI (http://www.ncbi.nim.nih.gov/Entrez/) and GenPept (http://www.ncbi.nlm.nih.gov/Entrez/protein.html) databases. Alternative databases for identifying protein sequences are known to the art and include, for example, Swissprot (http://www.expasy.ch/sprot/sprot-top.html), and owl (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/OWL/OWL.html.) The data set so obtained provides for the identification of proteins present in mitochondria from human heart, a bioenergetically active tissue.

[0109] As described in greater detail below, the present invention is also directed in pertinent part to the use of mass spectrometry (MS), and in particular to the use of matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, for the analysis of mitochondrial proteins and peptides obtained from a subject or biological source as provided herein.

[0110] In particularly preferred embodiments of the present invention, all or a portion of a protein fraction derived from a biological sample as provided herein may be contacted with one or more proteolytic agents under conditions and for a time sufficient to generate a plurality of peptide fragments derived from the protein fraction. Peptide fragments are typically continuous portions of a polypeptide chain derived from a protein of the protein fraction, which portions may be up to about 100 amino acids in length, preferably up to about 50 amino acids in length, more preferably up to about 30 amino acids in length, and still more preferably up to about 15-20 amino acids in length. In particularly preferred embodiments peptide fragments are 10-15 amino acids in length, and in other preferred embodiments peptide fragments may be 2-12 amino acids long.

[0111] A variety of proteolytic agents and suitable conditions for using them are known in the art, any of which may be useful according to certain embodiments of the present invention wherein peptide fragments are generated. Particularly preferred are proteolytic agents that are proteolytic enzymes or proteases, for example trypsin, Glu-C protease (Staphylococcal V8 protease), Lys-C protease, Arg-C protease, or other proteases known in the art to cleave peptides at specific amino acid linkages, typically at a relatively limited number of cleavage sites within a protein or polypeptide. Other useful proteolytic agents that are proteolytic enzymes include serine proteases, for example, chymotrypsin, elastase and trypsin; thiolproteases, such as papain or yeast proteinase B; acid proteases, including, e.g., pepsin or cathepsin D; metalloproteinases (e.g., collagenases, microbial neutral proteinases); carboxypeptidases; N-terminal peptidases or any other proteolytic enzymes that those having ordinary skill in the art will recognize may be employed to generate peptide fragments as provided herein (see, e.g., Bell, J. E. and Bell, E. T., Proteins and Enzymes, 1988 Prentice-Hall, Englewood Cliffs, N.J.; Worthington Enzyme Manual, V. Worthington, ed., 1993 Worthington Biochemical Corp., Freehold, N.J.).

[0112] Alternatively, in certain embodiments it may be desirable to use proteolytic agents that are chemical agents, for example HCl, CNBr, formic acid, N-bromosuccinimide, BNPS-skatole, o-iodosobenzoic acid/p-cresol, Cyssor, 2-nitro-5-thiocyanobenzoic acid, hydroxylamine, pyridine/acetic acid or other chemical cleavage procedures (see, e.g., Bell and Bell, 1988, and references cited therein).

[0113] As noted above, oxidative damage to proteins, such as protein modification that results from reactive free radical activity in biological systems, is an underlying feature in the pathogenesis of a number of diseases. Accordingly, a disease associated with altered mitochondrial function, for example a disease associated with altered mitochondrial constitution or composition (e.g., a disorder or condition characterized by statistically significant alterations in the quantity, structure and/or activity of one or more mitochondrial molecular components as provided herein) may also include a “disease associated with oxidative modification of a protein”, such as any disease in which at least one protein or peptide is oxidatively (e.g., covalently) and, in most cases, inappropriately modified. In highly preferred embodiments, at least one protein or peptide in a subject or biological source having a disease associated with oxidative modification of a protein includes a mitochondrial protein that has undergone disease-associated oxidative damage. Thus, such a disease may have a basis in a respiratory or metabolic or other defect, whether mitochondrial or extramitochondrial in origin. Diseases associated with oxidative modification of proteins may include Alzheimer's disease (AD), diabetes mellitus, Parkinson's disease, amyotrophic lateral sclerosis (ALS), atherosclerosis and other degenerative and inflammatory diseases. Those familiar with the art will be aware of clinical criteria for diagnosing certain of these diseases, which diagnostic criteria are augmented in view of the subject invention methods and compositions.

[0114] As described in greater detail in the Examples, certain embodiments of the invention contemplate the unexpected discovery that a mitochondrial protein or peptide containing tryptophan may be oxidatively modified to yield proteins or peptides containing this modified amino acid, although the invention is not intended to be so limited and as described herein contemplates mitochondrial proteins and peptides comprising a wide variety of other amino acids that may be oxidatively modified, according to oxidation reactions such as those described, for example, in Halliwell and Gutteridge (Free Radicals in Biology and Medicine, 1989 Clarendon Press, Oxford, UK). As described below, a number of mitochondrial proteins have been identified in which at least one tryptophan residue was doubly oxidized, thereby undergoing conversion to N-formylkynurenine. Accordingly, in certain embodiments the invention contemplates determination of a modified polypeptide (e.g., SEQ ID NOS:1-3025) comprising an oxidative modification that may, in certain further embodiments comprise an oxidized trytophan residue, which may in certain still further comprise N-formylkynurenine. Identification and determination of oxidative modification of tryptophan in proteins and peptides are well known to those familiar with the art (e.g., Halliwell and Gutteridge, pages 93-97; 315-320; 413-429).

[0115] For instance, the oxidation of tryptophan to N-formylkynurenine in proteins has been known for over 35 years since Previero et al. described it in hen's egg-white lysozyme in anhydrous formic acid (1967 J. Mol. Biol. 24:261). Kuroda et al. (1975 J. Biochem. (Tokyo) 78:641) subsequently found inactivation of lysozyme by ozone in aqueous solution occurred only when one critical tryptophan residue was oxidized, thus providing the first evidence that oxidation of a specific tryptophan residue can impair enzyme function. These early reports relied on identification of the tryptophan oxidation products by characteristic electronic absorption spectra. Finley et al. (1998 Protein Sci. 7:2391) exposed α-crystallin from bovine lens tissue to Fenton chemistry in vitro and separated the component tryptic peptides by HPLC. Tandem MS/MS spectrometry was used to identify oxidized amino acid sites by +16, +32 and +4 u increases in the molecular mass of peptide fragment ions containing tryptophan residues. Structures corresponding to those mass shifts are shown in FIG. 3. More recently Thiede et al. (2000 Rapid Commun. Mass Spectrom. 14:496) described oxidatively modified tryptophan residues in peptides from human Jurkat T lymphoblastoid cells. These workers described oxidatively modified tryptophan in a peptide which, as shown by the Examples provided herein, shares structure with a similar peptide derived from the mitochondrial voltage dependent anion channel-1 (VDAC1, e.g., SEQ ID NO:2559) polypeptide (see Table 3, KLETAVNLAWTAGNSNTR). Certain embodiments of the present invention therefore contemplate expressly excluding determination of the peptide KLETAVNLAWTAGNSNTR which comprises oxidatively modified tryptophan, certain other embodiments contemplate expressly excluding an oxidatively modified VDAC1 polypeptide, and certain other embodiments of the present invention therefore contemplate expressly excluding a disease associated with altered mitochondrial function that is T-cell lymphoma or leukemia.

[0116] In order to determine whether a mitochondrial component may contribute to a particular disease associated with oxidative modification of a protein, it may be useful to construct a model system for diagnostic tests and for screening candidate therapeutic agents in which the nuclear genetic background may be held constant while the mitochondrial genome is modified. It is known in the art to deplete mitochondrial DNA from cultured cells to produce η0 cells, thereby preventing expression and replication of mitochondrial genes and inactivating mitochondrial function. It is further known in the art to repopulate such η0 cells with mitochondria derived from foreign cells in order to assess the contribution of the donor mitochondrial genotype to the respiratory phenotype of the recipient cells. Such cytoplasmic hybrid cells, containing genomic and mitochondrial DNAs of differing biological origins, are known as cybrids. See, for example, International Publication Number WO 95/26973 and U.S. Pat. No. 5,888,498 which are hereby incorporated by reference in their entireties, and references cited therein.

[0117] According to the present invention, a level of at least one mitochondrial protein or peptide is determined in a biological sample from a subject or biological source. For subjects that are asymptomatic, that exhibit a pre-disease phenotype or that meet clinical criteria for having or being at risk for having a particular disease, such determination may have prognostic and/or diagnostic usefulness. For example, where other clinical indicators of a given disease are known, levels of at least one mitochondrial protein or peptide in subjects known to be free of a risk or presence of such disease based on the absence of these indicators may be determined to establish a control range for such level(s). The levels may also be determined in biological samples obtained from subjects suspected of having or being at risk for having the disease, and compared to the control range determined in disease free subjects. Those having familiarity with the art will appreciate that there may be any number of variations on the particular subjects, biological sources and bases for comparing levels of at least one mitochondrial protein or peptide that are useful. beyond those that are expressly presented herein, and these additional uses are within the scope and spirit of the invention.

[0118] For instance, determination of levels of at least one mitochondrial protein or peptide may take the form of a prognostic or a diagnostic assay performed on a skeletal muscle biopsy, on whole blood collected from a subject by routine venous blood draw, on buffy coat cells prepared from blood or on biological samples that are other cells, organs or tissue from a subject. Alternatively, in certain situations it may be desirable to construct cybrid cell lines using mitochondria from either control subjects or subjects suspected of being at risk for a particular disease associated with oxidative modification of proteins. Such cybrids may be used to determine levels of at least one mitochondrial peptide or protein for diagnostic or predictive purposes, or as biological sources for screening assays to identify agents that may be suitable for treating the disease based on their ability to alter (e.g., to increase or decrease in a statistically significant manner) the levels of at least one mitochondrial protein or peptide in treated cells.

[0119] In one embodiment of this aspect of the invention, therapeutic agents or combinations of agents that are tailored to effectively treat an individual patient's particular disease may be identified by routine screening of candidate agents on cybrid cells constructed with the patient's mitochondria. In another embodiment, a method for identifying subtypes of the particular disease is provided, for example, based on differential effects of individual candidate agents on cybrid cells constructed using mitochondria from different subjects diagnosed with the same disease.

[0120] MALDI

[0121] As noted above, in certain preferred embodiments of the present invention there is provided a method for identifying at least one mitochondrial protein comprising generating a mass spectrum of a mitochondrial polypeptide-derived peptide fragment, wherein the mass spectrum is preferably generated using MALDI-TOF. By way of background, in 1987, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) was introduced by Hillenkamp and Karas, and since has become a very powerful bioanalytical tool (Anal. Chem. 60:2288-2301,1988; see also Burlingame et al., Anal. Chem. 68:599-651, 1996 and references cited therein). The success of MALDI in the area of protein science can be attributed to several factors. The greatest of these is that MALDI can be rapidly (˜5 minutes) applied as an analytical technique to analyze small quantities of virtually any protein (practical sensitivities of ˜1 pmole protein loaded into the mass spectrometer). The technique is also extremely accurate. Beavis and Chait demonstrated that the molecular weights of peptides and proteins can be determined to within ˜0.01% by using methods in which internal mass calibrants (x-axis calibration) are introduced into the analysis (Anal. Chem. 62:1836-40, 1990). MALDI can also be made quantitative using a similar method in which internal reference standards are introduced into the analysis for ion signal normalization (y-axis calibration). Quantitative determination of proteins and peptides is possible using this approach with accuracies on the order of ˜10% (Nelson et al., Anal. Chem. 66:1408-15, 1994). Finally, MALDI is extremely tolerant of large molar excesses of buffer salts and, more importantly, the presence of other proteins.

[0122] With the high tolerance towards buffer salts and other biomolecular components comes the ability to directly analyze complex biological mixtures. Many examples exist where MALDI is used to directly analyze the results of proteolytic or chemical digestion of polypeptides (see Burlingame et al., supra). Other examples extend to elucidating post-translational modifications (namely carbohydrate type and content), a process requiring the simultaneous analysis of components present in a heterogeneous glycoprotein mixture (Sutton et al., Techniques in Protein Chemistry III, Angeletti, Ed., Academic Press, Inc., New York, pp.109-116,1993). Arguably, the most impressive use of direct mixture analysis is the screening of natural biological fluids. In that application, proteins are identified, as prepared directly from the host fluid, by detection at precise and characteristic mass-to-charge (m/z) values (Tempst et al., Mass Spectrometry in the Biological Sciences, Burlingame and Carr, Ed., Humana Press, Totowa, N.J., p.105, 1996).

[0123] The use of an affinity ligand-derivatized support to selectively retrieve a target analyte specifically for MALDI analysis was first demonstrated by Hutchens and Yip (Rapid Commun. Mass Spectrom. 7:576-80, 1993). Those investigators used single-stranded DNA-derivatized agarose beads to selectively retrieve a protein, lactoferrin, from pre-term infant urine by incubating the beads with urine. The agarose beads were then treated as the MALDI analyte—a process involving mixing with a solution-phase MALDI matrix followed by deposition of the mixture on a mass spectrometer probe. MALDI then proceeded in the usual manner. Results indicated that the derivatized beads selectively retrieved and concentrated the lactoferrin; enough so to enable ion signal in the MALDI mass spectrum adequate to unambiguously identify the analyte at the appropriate m/z value (81,000 Da). A number of variations on this approach have since been reported. These include the use of immunoaffinity precipitation for the MALDI analysis of transferrins in serum (Nakanishi et al., Biol. Mass Spectrom. 23:230-33,1994), screening of ascites for the production of monoclonal antibodies (Papac et al., Anal. Chem. 66:2609-13, 1994), and the identification of linear epitope regions within an antigen (Zhao et al., Anal. Chem. 66:3723-26,1994). Even more recently, the affinity capture approaches have been made rigorously quantitative by incorporating mass-shifted variants of the analyte into the analysis (Nelson et al. Anal. Chem. 67:1153-58,1995). The variants are retained throughout the analysis (in the same manner as the true analyte) and observed as unique (resolved) signals in the MALDI mass spectrum. Quantification of the analyte is performed by equating the relative ion signals of the analyte and variant to an analyte concentration.

[0124] Suitable mass spectrometers include, but are not limited to, a magnetic sector mass spectrometer, a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, a quadrupole (rods or ion trap) mass spectrometer and a time-of-flight (TOF) mass spectrometer, and/or various hybrid instruments comprising combinations of any two or more of such types of mass analyzer (e.g., quadrupole/orthogonal TOF, Qq/TOF, TOF/TOF, etc.). In a preferred embodiment, the mass spectrometer is a time TOF mass spectrometer.

[0125] Since large molecules, such as peptides and proteins, are generally too large to be desorbed/ionized intact, a matrix is used to assist laser desorption/ionization of the same. This technique is referred to as matrix assisted laser desorption/ionization or (MALDI), and the matrix agent is referred to as a “MALDI matrix.” In short, the analyte is contacted with a suitable MALDI matrix and allowed to crystallize. Suitable MALDI matrix materials are known to those skilled in this field, and include, for example, derivatives of cinnamic acid such as α-cyano-4-hydroxycinnamic acid (ACCA) and sinapinic acid (SA).

[0126] A first criterion to performing mass spectrometry on the analyte captured by the interactive surface is the generation of vapor-phase ions. In the practice of this invention, such species are generated by desorption/ionization techniques. Suitable techniques include desorption/ionization methods derived from impact of particles with the sample. These methods include fast atom bombardment (FAB—impact of neutrals with a sample suspended in a volatile matrix), secondary ion mass spectrometry (SIMS—impact of keV primary ions generating secondary ions from a surface), liquid SIMS (LSIMS—like FAB except the primary species is an ion), plasma desorption mass spectrometry (like SIMS except using MeV primary ions), massive cluster impact (MCI—like SIMS using large cluster primary ions), laser desorption/ionization (LDI—laser light is used to desorb/ionize species from a surface), and matrix-assisted laser desorption/ionization (MALDI—like LDI except the species are desorbed/ionized from a matrix capable of assisting in the desorption and ionization events). Any of the aforementioned desorption/ionization techniques may be employed in the practice of the present invention. In a preferred embodiment, LDI is employed, and in a more preferred embodiment, MALDI is utilized. For matrix assisted laser desorption ionization/time of flight (MALDI-TOF) analysis or other MS (mass spectrometry) techniques known to those skilled in the art, see, for example, U.S. Pat. Nos. 5,622,824, 5,605,798 and 5,547,835. Alternatively, other soft-ionization mechanisms that are not based on particle bombardment but that are also capable of ionizing peptides and/or proteins could be employed. Such methods include electrospray ionization (ESI, liquid flow containing analyte sprayed from a nozzle or needle at high voltage) or atmospheric pressure ionzation (API).

[0127] Screening Assays and Agents

[0128] In certain embodiments, the present invention provides a method of identifying an agent for treating a disease associated with altered mitochondrial function, comprising (a) contacting a candidate agent with a biological sample from a subject having a disease associated with altered mitochondrial function, wherein the sample comprises at least one polypeptide that exhibits altered biological activity which accompanies the disease and wherein the polypeptide is (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, or (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025; and (b) determining an increase or decrease in the altered biological activity of the polypeptide in the presence of the candidate agent relative to the level-of the altered biological activity in the absence of the candidate agent, and therefrom identifying an agent for treating a disease associated with altered mitochondrial function.

[0129] Candidate agents for use in these and related methods of screening for a modulator of mitochondrial protein or peptide according to the present invention may be provided as “libraries” or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as “small molecules” and having molecular weights less than 105 daltons, preferably less than 104 daltons and still more preferably less than 103 daltons. For example, members of a library of test compounds can be administered to a plurality of samples, and then assayed for their ability to increase or decrease the level of at least one indicator of altered mitochondrial function.

[0130] Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels. For example, various starting compounds may be prepared employing one or more of solid-phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions. The resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694, PCT/US91/04666, which are hereby incorporated by reference in their entireties) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No. 5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629, which are hereby incorporated by reference in their entireties). Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures, and tested for their influence on an indicator of altered mitochondrial function, according to the present disclosure.

[0131] The present invention provides compositions and methods that are useful in pharmacogenomics, for the classification and/or stratification of a subject or patient population. In one embodiment, for example, such stratification may be achieved by identification in a subject or patient population of one or more distinct profiles of at least one mitochondrial protein or peptide that is modified (e.g., an altered expression level, altered amino acid sequence, altered posttranslational modification or an oxidative modification) or in which the biological activity is altered and that correlates with a particular disease associated with altered mitochondrial function. Such profiles may define parameters indicative of a subject's predisposition to develop the particular disease, and may further be useful in the identification of novel subtypes of that disease. In another embodiment, correlation of one or more traits in a subject with at least one mitochondrial protein or peptide (e.g., expression levels of a mitochondrial protein that can be determined to differ from a control in a statistically significant manner) may be used to gauge the subject's responsiveness to, or the efficacy of, a particular therapeutic treatment. Similarly, where levels of at least one indicator mitochondrial protein or peptide and risk for a particular disease associated with altered mitochondrial function are correlated, the present invention provides advantageous methods for identifying agents suitable for treating such disease(s), where such agents affect levels of at least one mitochondrial protein or peptide (or levels of a modification) in a biological source. Such suitable agents will be those that alter (e.g., increase or decrease) the level of at least one mitochondrial protein or peptide in a statistically significant manner. In certain preferred embodiments, a suitable agent alters a mitochondrial protein or peptide level in a manner that confers a clinical benefit, and in certain other, non-exclusive preferred embodiments, a suitable agent alters a mitochondrial protein or peptide level by causing it to return to a level detected in control or normal (e.g., disease-free) subjects.

[0132] As described herein, determination of levels of at least one mitochondrial protein or peptide may also be used to stratify a patient population (i.e., a population classified as having one or more diseases associated with altered mitochondrial function, for example, by oxidative modification of a protein). Accordingly, in another preferred embodiment of the invention, determination of levels of a mitochondrial protein or peptide in at least one protein or peptide in a biological sample from an oxidatively stressed subject may provide a useful correlative indicator for that subject. A subject so classified on the basis of mitochondrial protein expression levels may be monitored using any known clinical parameters for a specific disease referred to above, such that correlation between levels of at least one mitochondrial protein or peptide and any particular clinical score used to evaluate a particular disease may be monitored. For example, stratification of an AD patient population according to levels of at least one mitochondrial protein or peptide may provide a useful marker with which to correlate the efficacy of any candidate therapeutic agent being used in AD subjects.

[0133] In certain other embodiments, the invention provides a method of treating a patient having a disease associated with altered mitochondrial function by administering to the patient an agent that that compensates for at least one biological activity of a polypeptide that exhibits altered biological activity which accompanies the disease, wherein the polypeptide is (i) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025, or (ii) a modified polypeptide that comprises at least one modification to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOS 1-3025. As known to the art, an agent that “compensates” for an altered biological activity of a polypeptide includes an agent that counterbalances any structural or functional defect or alteration in such polypeptide, such as an altered biological activity arising as the result of a modification as provided herein, where such counterbalancing may be partial or full restoration of normal activity, or restoration to supranormal levels, so long as an effect is demonstrable in a statistically significant manner. In certain preferred embodiments the agent substantially restores at least one mitochondrial protein or peptide to a level found in control or normal subjects (which in some cases may be an undetectable level). In a most preferred embodiment, an agent that substantially restores (e.g., increases or decreases) at least one mitochondrial protein or peptide to a normal level effects the return of the level of that indicator to a level found in control subjects. In another preferred embodiment, the agent that substantially restores such an indicator confers a clinically beneficial effect on the subject. In another embodiment, the agent that substantially restores the indicator promotes a statistically significant change in the level of at least one mitochondrial protein or peptide. As noted herein, those having ordinary skill in the art can readily determine whether a change in the level of a particular mitochondrial protein or peptide brings that level closer to a normal value and/or clinically benefits the subject, based on the present disclosure. Thus, an agent that substantially restores at least one mitochondrial protein or peptide to a normal level may include an agent capable of fully or partially restoring such level. These and related advantages will be appreciated by those familiar with the art.

[0134] Any of the agents for treating a disease associated with altered mitochondrial function (e.g., oxidative modification of a protein), identified as described herein, are preferably part of a pharmaceutical composition when used in the methods of the present invention. The pharmaceutical composition will include at least one of a pharmaceutically acceptable carrier, diluent or excipient, in addition to one or more agents for treating a disease associated with oxidative modification of a protein, and, optionally, other components.

[0135] “Pharmaceutically acceptable carriers” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.

[0136] “Pharmaceutically acceptable salt” refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

[0137] The pharmaceutical compositions that contain one or more agents for treating a disease associated with oxidative modification of a protein may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e.g., sublingually or buccally), sublingual, rectal, vaginal, intrathecal and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrameatal, intraurethral injection or infusion techniques. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.

[0138] For oral administration, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose. Coloring and/or flavoring agents may be present. A coating shell may be employed.

[0139] The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to one or more agents for treating a disease associated with oxidative modification of a protein, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

[0140] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0141] A liquid composition intended for either parenteral or oral administration should contain an amount of agent(s) for treating a disease associated with oxidative modification of a protein such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 wt % of an agent for treating a disease associated with oxidative modification of a protein in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral compositions contain between about 4% and about 50% of the agent for treating a disease associated with oxidative modification of a protein. Preferred compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active compound.

[0142] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the agent(s) for treating a disease associated with oxidative modification of a protein of from about 0.1 to about 10% w/v (weight per unit volume).

[0143] The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

[0144] In the methods of the invention, the agent(s) for treating a disease associated with oxidative modification of a protein may be administered through use of insert(s), bead(s), timed-release formulation(s), patch(es) or fast-release formulation(s).

[0145] It will be evident to those of ordinary skill in the art that the optimal dosage of the agent(s) for treating a disease associated with oxidative modification of a protein may depend on the weight and physical condition of the patient; on the severity and longevity of the physical condition being treated; on the particular form of the active ingredient, the manner of administration and the composition employed. It is to be understood that use of an agent for treating a disease associated with oxidative modification of a protein in a chemotherapy can involve such a compound being bound to an agent, for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound.

[0146] These and related advantages will be appreciated by those familiar with the art. The following Examples are offered by way of illustration and not limitation.

EXAMPLES

Example 1

PREPARATION OF HUMAN HEART MITOCHONDRIA

[0147] Human heart mitochondria were obtained from Analytical Biological Services (Wilmington, Del.) and were further purified by metrizamide gradient centrifugation (see, e.g., Rosenthal, R. E., et al., 1987, J. Cereb. Blood Flow Metab. 7:752-8). Mitochondria (40 mg) were resuspended in MSHE (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, 1 mM EGTA plus a Complete protease inhibitor cocktail tablet (Roche, Indianapolis, Ind.)) and loaded onto a 35%/17% metrizamide gradient in 6% Percoll. Gradients were centrifuged for 45 min at 19000 rpm, 4° C. in a SW40 rotor. The heavy mitochondrial fraction was collected from the 35/17% interface, diluted in MSHE before pelleting at 12000 g for 10 min, and resuspended in MSHE. Protein concentrations were determined using the BioRad DC protein assay (BioRad Laboratories, Hercules, Calif.). The purity of the mitochondria was assessed by Western analysis using antisera directed against actin (Abcam, Cambridge, UK), dynamin II (Transduction Labs, Lexington, Ky.), KDEL, and LAMP1 (Stressgen, Victoria, BC Canada) to detect contamination due to cytoplasm, plasma membrane, ER, and lysosomes, respectively. The integrity of the mitochondria was assessed by Western analysis using a cocktail of antibodies directed against components of the electron transport chain; NDUFS2, 70 kD subunit of complex II, core I of complex III, cox 4, and ATP synthase alpha; all from Molecular Probes (Eugene, Oreg.). A representative example of western immunoblot analysis of mitochondrial fractions prepared essentially as described here is shown in FIG. 1.

Example 2

SUCROSE DENSITY GRADIENT FRACTIONATION OF SOLUBILIZED MITOCHONDRIA

[0148] Metrizamide purified mitochondria (13 mg) were resuspended in MSHE plus protease inhibitors and solubilized with 1% lauryl maltoside for 25 min on ice with frequent vortexing. Samples were centrifuged at 14000 rpm, 4° C. for 20 min. The pellet was frozen by immersion in liquid nitrogen and stored at −80° C. The supernatant was subjected to sucrose gradient centrifugation (Hanson, B. J. et al., 2001, Electrophoresis 22:950-959). The gradient consisted of 1 mL step-fractions of 35, 32.5, 30, 27.5, 25, 22.5, 20, 17.5, 15 and 10% sucrose in 10 mM Tris, pH 7.5/1 mM EDTA/0.05% lauryl maltoside, plus protease inhibitors). The solubilized mitochondria were loaded onto the gradient in 5% sucrose and centrifuged at 38000 rpm, 4° C. for 16.5 h in a SW40 rotor. The gradient was collected from the bottom in 1 mL fractions. The gradient fractions were concentrated in Microcon YM-3 centrifugal concentrators (Millipore, Bedford, Mass.). The concentrated samples were quantitated using the BioRad DC protein reagent, snap frozen by immersion in liquid nitrogen and stored at −80° C. Separation of proteins across the gradient was initially assessed by subjecting 1 □L aliquots of the concentrated fractions to electrophoresis on precast 4-12% NuPAGE gels in Mes buffer (Invitrogen, Carlsbad, Calif.) followed by staining with SimplyBlue Safe Stain (Invitrogen) or Western analysis using the cocktail of antibodies directed against components of the electron transport chain. Quantification of the electron transport chain complexes across the gradient was performed on images captured on a Fluor-S Multilmager (BioRad, Hercules, Calif.) and analyzed using QuantityOne software (BioRad).

[0149] Immediately prior to processing and analysis by mass spectrometry (see below), the concentrated gradient fractions and the solubilized pellet were successively subjected to electrophoresis on NuPAGE gels using ultraclean reagents. Buffers were made using HPLC grade water, and a gel rig and staining box were set aside for these samples. Aliquots (25 μg) of each concentrated gradient fraction were loaded on a 4-12% NuPage gel and run at 25 mA for 1 h, then 35 mA for another 1 h 20 min. Gels were fixed for 10 min (40% methanol, 10% acetic acid), washed three times for 5 min in HPLC grade water, stained with colloidal Coomassie for 10-15 sec, and then partially destained in water.

Example 3

GEL PROCESSING AND MASS SPECTROMETRIC ANALYSIS OF POLYPEPTIDES

[0150] The lightly Coomassie-stained electrophoretic gels from Example 2 were imaged placed on a light box in a laminar flow hood on a plastic cutting mat with a 65×1 mm grid placed underneath. To avoid keratin contamination all manipulations were performed wearing latex gloves, shower caps and lab coats. Starting at the bottom the gel, approximately 1 mm slices were excised across the entire width of a gel lane with a clean razor, further cut into approximately 1 mm cubes and transferred to 500 μL microcentrifuge tubes that had been prewashed with 50:50 water:acetonitrile. This procedure was progressively continued to the top the gel to ensure comprehensive coverage of all proteins in the gel lane. Although most gel slices were 1 mm thick, when discrete bands were encountered they were selectively excised, while near the top of the gel slightly thicker slices were taken where the protein concentration was lower. This resulted in 50-64 slices for each of the 12 lanes processed (corresponding to sucrose fractions 1-10, combined 11/12 and the pellet).

[0151] The gel pieces were incubated with 200 μL destain solution (25 mM ammonium bicarbonate, 25% acetonitrile) at 37° C. for 45 min. The destain solution was decanted and another cycle of destaining performed if there was residual coloration. The gel pieces were then dried on a Genevac concentrator using the “cool heat” setting (about 30 min). The dried gel pieces were slightly moistened with 5 μL 50 mM ammonium bicarbonate, 5% acetonitrile and 5 μL of freshly prepared ice cold Promega modified trypsin (0.1 mg/mL in 50 mM ammonium bicarbonate, 5% acetonitrile) added. The gel pieces were allowed to soak up the trypsin solution for 10 min, and then were fully reswelled with a 65 μL aliquot of 50 mM ammonium bicarbonate, 5% acetonitrile. After an overnight incubation at 37° C., the digestion was terminated by addition of 7.5 μL 10% acetic acid followed by brief vortexing and light centrifugation in a microcentrifuge. The digest supernatants were subsequently transferred to secondary prewashed 500 μL microcentrifuge tubes and carefully concentrated using the Genevac to final volumes of 10-20 μL. At no stage were the digests taken to dryness, in order to avoid irreversible adsorption of low abundance peptides to the walls of the tubes.

[0152] The concentrated digests were then carefully decanted to avoid particulates and transferred to the wells of a V-bottom 220 μL polypropylene microtiter 96 well plate. This plate was directly placed in a Symbiot (Applied Biosystems, Foster City, Calif.) robotic MALDI target spotter and 0.5 μL aliquots were spotted on a 2×96 well PS1 MALDI target along with a 0.3 μL aliquot of alpha-hydroxycinnamic acid matrix in 50% ACN, 0.1% TFA. Between each row of sample spots, calibrant (Des Arg1 Bradykinin, Mr 904.4681; angiotensin 1, 1296.6853; Glul-Fibrinopeptide B, 1570.6774; Neurotensin, 1672.9175) was spotted for close external calibration between each successive MALDI spectrum.

[0153] MALDI spectra were acquired on a Voyager DE-STR under the following conditions: positive reflectron mode with delayed extraction, accelerating voltage 20 kV, grid voltage 65%, mirror voltage ratio 1.12, extraction delay time 125 nsec and low mass gate 500 Da. Spectral acquisition was automated using a spiral search pattern with saved spectra being the average of 3 successful acquisitions from 400 laser shots at 20 Hz repetition rate in the m/z 850-3000 range with a minimum intensity of 750 counts in the m/z 1000-3000 range. Peptide mass fingerprints were analyzed using the program Protein Prospector (Clauser, K. R. et al., 1999, Analytical Chemistry 71, 14:2871). Peaks from baseline corrected, noise filtered deisotoped spectra were filtered to remove autolytic trypsin and most keratin peaks and then subjected to two modes of analysis. The first involved tolerant matching of 4 or 5 peaks to proteins in the database within a 100 ppm window. In general, proteins matching with MOWSE scores (see Pappin, D. J. C. et al., 1993, Current Biology 3: 327-332 for an explanation of MOWSE scores) in excess of 10000 were considered hits. The second analysis involved using the program “intellical” (Applied Biosystems) which demands high precision. As a first pass, 25 proteins would be selected from the database with 3 matches with in 150 ppm mass accuracy. The program would then look for a uniform deviation between the observed and calculated peptide masses and recalibrate the spectrum against the best fits. In general, a protein was considered a hit that had 4 peptides matching within 15 ppm of the recalibrated spectrum and MOWSE scores over 1000 using these more rigorous parameters. These analyses were fully automated using PS1 software (Applied Biosystems). FIG. 2 shows a representative example of a MALDI mass spectrum generated from polypeptides derived from a single one-dimensional gel slice.

[0154] As well as these selection criteria, the relative intensity of the matching peaks and the molecular weight of the identified protein relative to the band from which it was excised were also taken into account. The remaining portions of the digests were subjected to automated LC/MS/MS analysis. The microtiter plate containing the remaining peptide digest mixture were transferred to an Endurance autosampler connected to a MicroTech Ultimate LC system. The digest (10 μL) was transferred to a capillary trapping column containing C18 reversed phase resin at 20 μL/min using a third pump containing solvent A (95% water, 5% acetonitrile, 0.5% acetic acid) and washed for 3 min. A gradient of solvent A to solvent B (80% acetonitrile, 20% water, 0.5% acetic acid) 20% to 80% over 40 min was used to elute peptides through a 4.5 cm 75 μC-18 packed Picofrit column (New Objectives Inc., Woburn, Mass.) at a flow rate of 200-500 nL/min directly into the heated capillary orifice of a Finnigan LCQ Ion Trap Mass spectrometer equipped with a Finnigan dynamic nanospray source (Thermo Finnigan, San Jose, Calif.).

[0155] Mass spectra were acquired in the m/z 400-2000 range under the following conditions: positive polarity, capillary temperature 148° C., source voltage 2.4 kV, source current 80 μA, capillary voltage 29 V and tube lens offset 0 V. After one full scan MS of the column effluent was recorded, two MS/MS spectra of the most intense and second most intense MS peaks were recorded over the m/z 100-2000 range with an isolation width of 2.5 and normalized collision energy 35. Dynamic exclusion was employed to select the maximum number of unique peptide peaks from the chromatograms. After replicate MS/MS spectra were acquired for a precursor ion, the m/z value of ion was placed on an exclusion list with a ±1.5 u window for 3 min. Each chromatogram was subsequently analyzed with the program SEQUEST (Ducret et al., 1998, Protein Sci. 7: 706-719). The minimum requirement for a hit were at least 2 peptides for a particular protein having an Xcorr>1.7 for a +1 ion, Xcorr>2 for a +2 ion or Xcorr>3. In all cases Δcorr must be greater than 0.1.

[0156] A set of 3025 polypeptides [SEQ ID NOS:1-3025] was identified in the GENBANK database on the basis of the above-described selection criteria for hits from the mitochondrial protein preparations recovered according to the procedures detailed above. Table 1 presents the numbers [SEQ ID NOS:1-3025] corresponding to the Sequence Listing submitted herewith for all 3025 polypeptides identified herein as mitochondrial components, along with the GENBANK accession numbers for these sequences and (if known) a brief description of each protein based on its sequence characteristics and database annotation. Additional polypeptides that were identified included those having amino acid sequences as set forth in NCBI/Genbank Acc. Nos. 35655 and 1421609, and reference herein to any one of SEQ ID NOS:1-3025 may according to certain embodiments be understood to include NCBI/Genbank Acc. Nos. 35655 and 142160.

1TABLE 1HUMAN HEART MITOCHONDRIAL PROTEINSSEQ IDGENBANKNO:ACC. NO.DESCRIPTION OF MITOCHONDRIAL PROTEINS113013ND 4228590reading frame HSA328714anion transport protein430102type I collagen531474follicle stimulating hormone receptor631645glyceraldehyde 3-phosphate dehydrogenase731746glutathione-insulin transhydrogenase (216 AA)834670hexokinase 1934719myeloperoxidase1072146vitronectin precursor - human1172222heat shock protein 90-beta - human1286754carrier ANT3 - human (fragment)1387528dnaK-type molecular chaperone HSPA5 precursor - human1488512protein-L-isoaspartate(D-aspartate) O-methyltransferase (EC 2.1.1.77)splice form II - human1588650succinate dehydrogenase (ubiquinone) (EC 1.3.5.1) 27K iron-sulfurprotein precursor, mitochondrial - human (fragment)1688741T-cell receptor beta chain V region - human (fragment)1788972undulin 118105294alternative splicing factor ASF-219105475myosin-binding protein C, skeletal muscle - human20105595cell adhesion protein SQM121106140glycophorin A22106185GTP-binding protein Rab223106906lipopolysaccharide-binding protein24106970mcf2 protein25107554pyruvate kinase isozyme M226107631ryanodine receptor type 1, skeletal muscle - human27107912transcription factor E328113962annexin VI29114312Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (Calcium pump 2)(SERCA2) (SR Ca(2+)-ATPase 2) (Calcium-transporting ATPasesarcoplasmic reticulum type, slow twitch skeletal muscle isoform)(Endoplasmic reticulum class 1/2 Ca(2+) ATPase)30114374Na, K-ATPase subunit alpha 131114374Sodium/potassium-transporting ATPase alpha-1 chain precursor (Sodiumpump 1) (Na+/K+ ATPase 1)32114549ATPase beta F133115206C-1-TETRAHYDROFOLATE SYNTHASE, CYTOPLASMIC (C1-THFSYNTHASE)34117103cox 5b35117759UCR 4 CYTOCHROME C136117863UCR cyt b37120643GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE, MUSCLE38120749MAJOR GASTROINTESTINAL TUMOR-ASSOCIATED PROTEINGA733-239121665Glutathione peroxidase 1 (GSHPx-1) (Cellular glutathione peroxidase)40123277HOMEOBOX PROTEIN HOX-C6(HHO.C8)41123571heat shock 27 KD protein42123678heat shock 90 kD protein HSP 90-ALPHA (HSP 86)43123678Heat shock protein HSP 90-alpha (HSP 86)44125484HEPATOCYTE GROWTH FACTOR RECEPTOR PRECURSOR(C-MET)(HGF-SF RECEPTOR)45129070pyruvate dehydrogenase E 1-beta46129379heat shock 60 kDa protein, mitochondrial precursor (Hsp60) (60 kDachaperonin) (CPN60) (Heat shock protein 60) (HSP-60) (Mitochondrialmatrix protein P1) (P60 lymphocyte protein) (HuCHA60)47129902Phosphoglycerate kinase 1 (Primer recognition protein 2) (PRP 2)48130749ALKALINE PHOSPHATASE, TISSUE-NONSPECIFIC ISOZYMEPRECURSOR49132164RETINOBLASTOMA-ASSOCIATED PROTEIN(P105-RB)50136066TRIOSEPHOSPHATE ISOMERASE51136090TROPOMYOSIN BETA CHAIN, SKELETAL MUSCLE52136213Troponin I, cardiac muscle53141686ZINC FINGER PROTEIN 854177836alpha-1-antitrypsin precursor55178345alloalbumin Venezia56178390aldehyde dehydrogenase57178426alpha-fodrin58178736apolipoprotein B10059178896beta-3-adrenergic receptor60179279ATPase beta subunit61180529chromogranin A62181238cytochrome c163184477retinoic acid receptor64188590myosin light chain 365188672mannose 6-phosphate receptor66189422proliferating cell nuclear protein P12067189514p80-coilin68190201porin69190474salivary proline-rich protein 170190804ubiquinone-binding protein71190804UCR 6 ubiquinone-binding protein72223374isomerase, triosephosphate73223582histone H474223632dismutase, Cu/Zn superoxide75224309protein delta T3, glyco76225897glycogen phosphorylase77225985amyloid related serum protein SAA78226007ventricular myosin L179226021growth regulated nuclear 68 protein80226209cox 881227297ND FeS NADH dehydrogenase FeS protein82227448phosphofructokinase83228097receptor-like Tyr phosphatase84229149hemoglobin beta85229479lipoprotein Gln I86229479lipoproteinGln I87230004Human Neutrophil Elastase (HNE) (E.C.3.4.21.37) (Also Referred To AsHuman Leucocyte Elastase (HLE)) Complex With Methoxysuccinyl-Ala-Ala-Pro-Ala Chloromethyl Ketone (MSACK)88231743G1/S-SPECIFIC CYCLIN D389232472nucleotide diphosphate kinase subunit A, p19/nm23-H1 [human, PeptidePartial, 12 aa, segment 1 of 3]90238427Porin 31HM [human, skeletal muscle membranes, Peptide, 282 aa]91251188protein phosphatase from PCR fragment H992283950oxoglutarate dehydrogenase (lipoamide) (EC 1.2.4.2) precursor - human93284319mucin-associated antigen - human (fragment)94285975rab GDI95292793T-cell receptor beta96306926insulin-like growth factor binding protein 297307021mu-immunoglobulin98312137aldolase C99337758pre-serum amyloid P component100338017SEF2-1D protein101339647thyroid hormone binding protein precursor102346275myelin transcription factor 1 - human (fragment)103352335reductase, NADH cytochrome b5104385479N-methyl-D-aspartate glutamate receptor channel; NMDA GluR channel105386745guanine nucleotide-binding protein G-s-alpha-3106386872myoglobin107387010pyruvate dehydrogenase E1-beta subunit precursor108387011pyruvate dehydrogenase E1-alpha109387011pyruvate dehydrogenase E1-alpha precursor110387016phosphoglycerate mutase111393124Unknown112416776CD27 LIGAND(CD70 ANTIGEN)113434755rat general mitochondrial matrix processing protease mRNA (RATMPP).,similar to114436222Unknown115438650paired box protein116448295TLS protein117458862heart fatty acid binding protein; hFABP118469045h-contactin 2 precursor119476780Ras guanine nucleotide exchange factor son-of-sevenless (sos) 1 -human120481043MHC class III histocompatibility antigen HLA-B-associated protein 2[similarity] - human121483239homeotic protein engrailed 2 - human122499158acetoacetyl-CoA thiolase mitochondrial123516764motor protein124516768motor protein125533538diamine oxidase, copper/topa quinone containing126551604pregnancy-specific beta-1 glycoprotein127553254cytochrome b5 reductase (EC 1.6.2.2)128553597myosin heavy chain beta-subunit129553734putative130553734Unknown131577307The ha3662 gene product is related to mouse glycerophosphatedehydrogenase.132595267gastrin-binding protein 78 kDa133606609GBP134627364adenovirus E1A-associated 130k protein - human135627367desmoyokin - human (fragments)136631070AHNAK-related protein - human (fragment)137687714dynein heavy chain, isotype 1B138703083cytochrome b5139704445ATPase subunit 8140728834Alu subfamily SB2 sequence contamination warning entry141802150pancreatic peptidylglycine alpha-amidating monooxygenase; PA142903598Krueppel-type zinc finger protein143992629orf1441000865This CDS feature is included to show the translation of the correspondingV_region. Presently translation qualifiers on V_region features are illegal1451001941dihydropyridine receptor alpha 1 subunit1461033182Y-chromosome RNA recognition motif protein1471053081calpastatin1481065362Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length Non-Myristoylated1491070477insulin receptor precursor - human1501071834dihydrolipoamide S-succinyltransferase1511082355epidermal autoantigen 450K (clone pE450-B) - human (fragment)1521082428GTPase-activating protein rhoGAP1531082553JC-kappa protein1541082567laminin A31551082692phospholipase C beta 31561082723propionyl Coenzyme A carboxylase, beta polypeptide1571082723propionyl-CoA carboxylase (EC 6.4.1.3) beta chain precursor - human1581085294cell-cycle-dependent 350K nuclear protein - human (fragment)1591085373protein disulfide-isomeraseER60 precursor1601091688heat shock protein1611096024isoAsp protein carboxyl methyltransferase1621096067tat-associated protein1631103677myosin-light-chain kinase1641124876Krueppel-related DNA-binding protein1651130694erythrocyte adducin alpha subunit1661136416mitosis-specific chromosome segregation protein SMC1 of S. cerevisiae.,similar to1671136741predicted protein of 548 amino acids1681151113PDE1C31691160932DRAL gene product gi|7209525|dbj|BAA92253.1| (AB038794)DRAL/Slim3/FHL21701168719C6.1A PROTEIN1711168781EXTRACELLULAR CALCIUM-SENSING RECEPTOR PRECURSOR1721169072APOPAIN PRECURSOR (CYSTEINE PROTEASE CPP32) (YAMAPROTEIN) (CPP-32) (CASPASE-3)1731169204dodecenoyl-CoA Delta-isomerase1741170654ANTIGEN KI-671751172554VDAC-21761174572Thromboxane A2 receptor (TXA2-R) (Prostanoid TP receptor)1771177230zinc finger1781177438brca21791184699tyrosyl-tRNA synthetase1801196398Unknown1811196433Unknown1821220311elongation factor-1 alpha1831235848HMG CoA synthase1841235902FRAP-related protein1851237406Cu/Zn-superoxide dismutase1861245894cardiac myosin binding protein-C1871245985beta 2-adrenergic receptor, beta 2AR {Y354A} [human, Peptide PartialMutagenesis, 24 aa]1881246236ptp-IV1b, PTP-IV1 gene product1891262579ND 11901262580ND 21911262581cox 11921262582ATPase 61931292941hydroxymethylglutaryl-CoA lyase1941293561Diff40 gene product1951335064fibrillin1961335072G34 (big gastrin)1971335212medullasin N-term.1981335250Rod cGMP phosphodiesterase1991335277Unknown2001340142alpha1-antichymotrypsin2011346317heat shock 70 kD protein 72021351900NEUROBLAST DIFFERENTIATION ASSOCIATED PROTEIN2031351900[Segment 1 of 2] Neuroblast differentiation associated protein AHNAK(Desmoyokin)2041351901NEUROBLAST DIFFERENTIATION ASSOCIATED PROTEIN2051354222aldehyde dehydrogenase E32061359715Na+, K+ ATPase2071359715Na+, K+ ATPase2081359759histamine H2 receptor2091362755endopeptidase La homolog (EC 3.4.21.—) precursor, mitochondrial(version 1)2101381814skeletal muscle LIM-protein SLIM2111399105phosphatidylinositol (4,5)bisphosphate 5-phosphatase homolog2121399801p1672131408188desmin2141504020Yeast translation activator GCN1 (P1: A48126), similar to2151517899RAGE-1 ORF5; one of 3 possible coding regions2161582692TATA box-binding protein2171587138sorcin2181587477TCOF1 gene2191588292Ca channel: SUBUNIT = alpha: ISOTYPE = L2201655594HES12211657266S10 GTP-binding protein2221665723RPD3 protein2231688267polo like kinase2241706611ELONGATION FACTOR TU, MITOCHONDRIAL PRECURSOR2251708098Histone H1t2261709123DNA MISMATCH REPAIR PROTEIN MSH6 (MUTS-ALPHA 160 KDASUBUNIT2271709947PYRUVATE CARBOXYLASE PRECURSOR2281710279dihyrolipoamide acetyl transferase2291718502aconitase mitochondrial2301718502aconitase, mitochondrial2311730078130 KDA LEUCINE-RICH PROTEIN(GP130)2321731414ZINC FINGER PROTEIN 1382331762533carnitine palmitoyltransferase I2341763238lysosomal trafficking regulator LYST2351773381APXL2361778410unknown2371778432Treacher Collins syndrome2381805280alpha II spectrin2391869803fatty acid binding protein 32401930110GM-CSF receptor alpha subunit soluble 32411942187Lactoferrin, H253m N Terminal Lobe Of Human2421943532Profilin I Crystallized In High Salt Actin-Binding Protein, Human Platelet24320783293-hydroxyacyl-CoA dehydrogenase, isoform 22442078470Putative gene. Genscan predictions confirmed by EST splicing.; coded forby human cDNAs AA122029 (NID: g1678048), D31562 (NID: g644442),AA158721 (NID: g1733515), R59640 (NID: g830335) and F13082(NID: g709111)2452114493RNA editase2462117022zinc finger 5 protein2472117163leukocyte antigen, HLA-A2 variant2482117707dihydrolipoamide S-(2-methylpropanoyl)transferase (EC 2.3.1.—) precursor -human2492117873pyruvate kinase (EC 2.7.1.40), muscle splice form M1 - human2502118344arginine - tRNA ligase (EC 6.1.1.19) - human2512118970histone H1 - human (fragment)2522119268alpha-tubulin - human (fragment)2532119390proapo-A-I protein - human2542119533giantin2552119712dnaK-type molecular chaperone HSPA1L heat shock protein2562119918P43 - human2572134903CG1 protein, kinectin 12582135068enhancer protein2592135611melanoma ubiquitous mutated protein - human (fragment)2602135819neuropolypeptide h3, brain26121359113′,5′-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) 4A, cAMP-specific, long splice form - human2622136207succinate-semialdehyde dehydrogenase (EC 1.2.1.24) - human(fragment)2632136282TOG protein2642144337pyruvate dehydrogenase (lipoamide) (EC 1.2.4.1) beta chain precursor,long splice form - human2652145011putative collagen homolog protein-b2662146960methyl CpG binding protein 2 - human (fragment)2672217933PKU-beta2682224581Unknown2692224583Unknown2702224621Unknown2712224663Unknown2722243110Unknown2732244654HS24/P522742270925beta4-integrin2752286145caspase-like apoptosis regulatory protein2762293556Ran binding protein 22772306809X-linked nuclear protein2782317769probable zinc finger protein H1012792393734C. elegans F11A10.5; 80% similarity to Z68297 (Pl2802393763NAD (H)-specific isocitrate dehydrogenase gamma subunit2812454586reverse transcriptase2822465178COX7RP2832498864RRP5 PROTEIN HOMOLOG2842499753PROTEIN-TYROSINE PHOSPHATASE KAPPA PRECURSOR2852506118MULTIDRUG RESISTANCE PROTEIN 12862507187PROTEIN-L-ISOASPARTATE(D-ASPARTATE) O-METHYLTRANSFERASE (PROTEIN-BETA-ASPARTATEMETHYLTRANSFERASE) (PIMT)2872511440calcium/calmodulin-dependent protein kinase II; CaM kinase II2882511779beta III spectrin2892565032transcription activator/repressor protein delta/YY1; similar2902624694Single-Stranded Dna Binding Protein, Human Mitochondrial2912653817lipopolysaccharide binding protein2922661211oxidative 3 alpha hydroxysteroid dehydrogenase2932662397HADHB2942665782voltage-gated sodium channel, subtype III2952695574leukocyte function-associated molecule-1 alpha subunit2962769254NIPSNAP2 protein2972769254NIPSNAP2 protein2982811135retinal rod Na+/Ca+, K+ exchanger2992822143R30217_13002852604Unknown3012865252Unknown3022873377exportin t3032981731Cypa Complexed With Hagpia3043012097F22329_13053021386zinc finger protein3063023143kappa 1 immunoglobulin light chain variable region3073043584Unknown3083043646Unknown3093046880LIM-homeodomain protein LMX1B/LMX1.23103114510T State Human Hemoglobin [alpha V96w], Alpha Aquomet, Beta Deoxy3113123721ND 24 K NADH dehydrogenase 24-kDa subunit of complex I3123153859thioredoxin delta 33133168604proline and glutamic acid rich nuclear protein isoform3143211975putative glialblastoma cell differentiation-related protein3153211977sarco-/endoplasmic reticulum Ca-ATPase 33163212539Isovaleryl-Coa Dehydrogenase At 2.6 Angstroms Resolution: StructuralBasis For Substrate Specificity3173252827Unknown3183252827Unknown3193256185target of myb1homolog)3203273228acyl-CoA dehydrogenase very-long-chain3213273386plasmalemmal porin3223294170dJ232K4.1 (hypothetical 141.7 kD protein JUMONJI)3233299887ES/130-related protein3243327040Unknown3253327054Unknown3263327054Unknown3273360457cul-33283402141Lysozymes At Constant Positions3293402145Lysozyme3303540239ND Fe-S2 NADH dehydrogenase-ubiquinone Fe-S protein 2 precursor3313599521musculin3323641621gp180-carboxypeptidase D-like enzyme3333641621gp180-carboxypeptidase D-like enzyme3343660040Fkbp Mutant F36v Complexed With Remodeled Synthetic Ligand3353660556hdkk-43363694663Unknown3373717965DIA-12C3383766197succinyl-CoA synthetase beta subunit, ATP-specific3393766197succinyl-CoA synthetase beta subunit, ATP-specific3403766199succinyl-CoA synthetase beta subunit GTP-specific3413766451CHRNB23423882147Unknown3433882301Unknown3443885362sepiapterin reductase3453891975Cathepsin G3463982589SOX-28 protein3473986482translation initiation factor elF3 p40 subunit; elF3p403484008131chaperonin 103494096860fibronectin3504097409PAX-93514103446NAD+-specific isocitrate dehydrogenase beta subunit isoform A3524127947guanine nucleotide-exchange factor3534139720Chymase3544151929PCAF-associated factor 4003554153874single-stranded mitochondrial DNA-binding protein precursor3564204963MUC-1/X mucin short variant3574206175ubiquitin-specific protease3584210351novel protein3594240227Unknown3604240243Unknown3614240305Unknown3624261577CD8 beta chain3634262430CMP-NeuAc: lactosylceramide alpha-2,3-sialyltransferase3644263556Unknown3654406346guanylate cyclase activating protein 33664406564succinyl-CoA synthetase beta subunit GTP-specific3674406651h-sco13684416457mitotic checkpoint protein3694495063yeast suppressor protein SRP40) dJ108K11.3 (similar to3704501869acyl-Coenzyme A oxidase 2, branched chain3714501967alpha-2C-adrenergic receptor; alpha-2C-1 adrenergic receptor; alpha-2C-1 adrenoceptor; alpha-2-adrenergic receptor, renal type; alpha2-AR-C43724502011adenylate kinase 13734502013adenylate kinase 2 isoform a; Adenylate kinase-2, mitochondrial3744502097solute carrier family 25 (mitochondrial carrier; adenine nucleotidetranslocator), member 4; adenine nucleotide translocator 1 (skeletalmuscle)3754502101annexin I3764502107annexin V3774502111annexin VII isoform 13784502201ADP-ribosylation factor 13794502273ATPase, Na+/K+ transporting, alpha 3 polypeptide3804502297ATPase delta F13814502303ATPase OSCP F13824502327AU RNA-binding protein/enoyl-Coenzyme A hydratase precursor3834502331arginine vasopressin receptor 1A; V1a vasopressin receptor;vascular/hepatic-type arginine vasopressin receptor; antidiuretic hormonereceptor 1A3844502379BCL103854502419biliverdin reductase B (flavin reductase (NADPH))3864502457ATP-binding cassette, sub-family B (MDR/TAP), member 11; ABCmember 16, MDR/TAP subfamily3874502459basigin; collagenase stimulatory factor; M6 antigen3884502509complement component 5 receptor 1 (C5a ligand); complementcomponent-5 receptor-2 (C5a ligand)3894502517carbonic anhydrase I3904502563calpain 2, large subunit3914502601carbonyl reductase 3; carbonyl reductase3 [Homo sap3924502603chromobox homolog 4 (Pc class homolog, Drosophila); chromoboxhomolog 4 (Drosophila Pc class)3934502703CDC6 homolog; CDC6 (cell division cycle 6, S. cerevisiae) homolog;CDC18 (cell division cycle 18, S. pombe, homolog)-like; CDC6-relatedprotein3944502719cadherin 13 preproprotein; H-cadherin; heart-cadherin; T-cad3954502841carbohydratesulfotransferase 13964502855sarcomeric mitochondrial creatine kinase precursor; creatine kinase,mitochondrial 2; basic-type mitochondrial creatine kinase3974502985cox 6b3984502987cox 7a muscle3994502989cox 7a liver4004502991cox 7b4014502993cox 7c4024503015copine III4034503021liver carnitine palmitoyltransferase I; L-CPT14044503049cysteine-rich protein 2; Cystein-rich intestinal protein4054503057crystallin, alpha B; crystallin, alpha-2; Rosenthal fiber component; heat-shock 20 kD like-protein4064503143cathepsin D4074503177chromosome X open reading frame 24084503269deoxycytidine kinase gi|11436224|ref|XP_0034740945033012,4-dienoyl CoA reductase 1 precursor4104503375dihydropyrimidinase4114503431dysferlin; dystrophy-associated fer-1-like 14124503443endothelin converting enzyme 14134503447peroxisomal enoyl-coenzyme A hydratase-like protein; delta3,5-delta2,4-dienoyl-CoA isomerase; peroxisomal enoyl-CoA hydratase 1; dienoyl-CoAisomerase4144503475eukaryotic translation elongation factor 1 alpha 24154503507eukaryotic translation initiation factor 2, subunit 34164503537eukaryotic translation initiation factor 4E binding protein 34174503607electron transfer flavoprotein alpha polypeptide4184503609electron transfer flavoprotein beta polypeptide4194503613envoplakin4204503651fatty-acid-Coenzyme A ligase, long-chain 14214503667fibrillin 2 + F4224224503731FK506-binding protein 64234503835frizzled 94244503843adaptor-related protein complex 1, gamma 2 subunit; gamma2-a4254503899N-acetylgalactosamine-6-sulfatase precursor4264503937glioblastoma amplified sequence4274504041guanine nucleotide binding protein (G protein), alpha inhibiting activitypolypeptide 2; Guanine nucleotide-binding protein (G protein), alpha-inhibiting4284504049guanine nucleotide binding proteintransducin alpha-chain4294504067aspartate aminotransferase 1; glutamic-oxaloacetic transamin4304504071platelet glycoprotein lb alpha polypeptide precursor4314504169glutathione synthetase4324504189glutathione transferase zeta 1 (maleylacetoacetate isomerase);glutathione transferase Zeta 14334504483hypoxanthine phosphoribosyltransferase 14344504487histidine-rich calcium-binding protein precursor SARCOPLASMICRETICULUM4354504517heat shock 27 kD protein 14364504521heat shock 60 kD protein 1 (chaperonin)4374504523heat shock 10 kD protein 1 (chaperonin 10)4384504523heat shock 10 kD protein 1 (chaperonin 10)4394504665interleukin 2 receptor, beta; Interleukin-2 receptor, beta polypeptide4404504689IMP (inosine monophosphate) dehydrogenase 24414504733insulin receptor substrate 44424504795inositol 1,4,5-triphosphate receptor, type 34434504867ring finger protein (C3HC4 type) 8; C3HC4-type zinc finger protein; zincfinger protein4444504975low density lipoprotein receptor precursor; LDLR precursor; LDL receptor4454504991leukemia inhibitory factor (cholinergic differentiation factor); cholinergicdifferentiation factor4464505071MAP-kinase activating death domain protein4474505093monoamine oxidase B4484505093monoamine oxidase B4494505145malic enzyme 2, NAD(+)-dependent, mitochondrial4504505145malic enzyme 2, NAD(+)-dependent, mitochondrial; Malic enzyme,mitochondrial; malic enzyme 2, mitochondrial; pyruvic-malic carboxylase;malate dehydrogenase4514505153MAP/ERK kinase kinase 34524505249mutS homolog 3 (E. coli); mutS (E. coli) homolog 34534505257moesin4544505257moesin4554505355ND B84564505357ND 9k NDUFA44574505359ND B144584505361ND B124594505363ND 16k, SGDH4604505365ND B174614505367ND 6k4624505369ND 18K NADH dehydrogenase (ubiquinone) Fe-S protein 4 (18 kD)(NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18 kD (NADH-coenzyme Q; mitochondrial respiratory chaincomplex I (18-KD subunit)4634505371ND 23K NADH dehydrogenase (ubiquinone) Fe-S protein 8 (23 kD)(NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone) Fe-S protein 8 (23 kD) (NADH-coenzyme Q4644505375neogenin homolog 1 (chicken); neogenin (chicken) homolog 14654505399NIPSNAP homolog 1; 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like 14664505405glycoprotein (transmembrane) nmb; transmembrane glycoprotein4674505591peroxiredoxin 1; Proliferation-associated gene A; proliferation-associatedgene A (natural killer-enhancing factor A)4684505621prostatic binding protein; phosphatidylethanolamine binding protein4694505685pyruvate dehydrogenase (lipoamide) alpha 1; Pyruvate dehydrogenase,E1-alpha polypeptide-14704505687pyruvate dehydrogenase (lipoamide) beta; Pyruvate dehydrogenase, E1beta polypeptide4714505693pyruvate dehydrogenase kinase, isoenzyme 44724505717peroxisomal biogenesis factor 11A4734505773Prohibitin4744505775carrier phosphate isoform B4754505775phosphate carrier precursor isoform 1b; phosphate carrier, mitochondrial;phosphate carrier, mitochondrial precursor4764505801phosphoinositide-3-kinase, class 34774505869phospholipase C, gamma 1 (formerly subtype 148)4784505887phospholamban4794505893proteolipid protein 24804505909peripheral myelin protein 2; M-FABP4814505911postmeiotic segregation 1; Postmeiotic segregation increased (S.cerevisiae)-like 14824505925putative neurotransmitter receptor4834505965POU domain, class 4, transcription factor 34844506077protein kinase C substrate 80 KD-H4854506091mitogen-activated protein kinase 64864506189proteasome (prosome, macropain) subunit, alpha type, 74874506197proteasome (prosome, macropain) subunit, beta type, 3; Proteasomesubunit, beta type, 34884506291protein tyrosine phosphatase, non-receptor type 2, isoform 1; T-cellprotein tyrosine phosphatase4894506371RAB5B, member RAS oncogene family4904506401raf proto-oncogene serine/threonine protein kinase4914506413RAP1A, member of RAS oncogene family; RAS-related protein RAP1A4924506445RNA binding motif protein 44934506517regulator of G-protein signalling 2, 24 kD4944506787IQ motif containing GTPase activating protein 1; rasGAP-like with IQmotifs4954506959TAL1 (SCL) interrupting locus; SCL interrupting locus4964506975carrier family 12 (sodium/potassium/chloride transporters), member 24974506977carrier family 12 (sodium/chloride transporters), member 34984506997solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier),member 11; solute carrier family 20 (oxoglutarate carrier), member 44994507007carrier family 25 (mitochondrial carrier, Aralar), member 12; calciumbinding mitochondrial carrier superfamily member Aralar5004507021solute carrier family 4, anion exchanger, member 1 (erythrocytemembrane protein band 3, Diego blood group)5014507185sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase);Sepiapterin reductase5024507215signal recognition particle 54 kD5034507299sudD suppressor of bimD6 homolog (A. nidulans); human homolog ofAspergillus nidulans sudD gene product; sudD (suppressor of bimD6,Aspergillus nidulans) homolog5044507389elongin A; transcription elongation factor B (SIII)5054507401transcription factor 6-like 15064507401transcription factor 6-like 1 (mitochondrial transcription factor 1-like)5074507431thyrotrophic embryonic factor; Thyrotroph embryonic factor5084507443transcription factor AP-2 beta (activating enhancer binding protein 2 beta);transcription factor AP-2 beta (activating enhancer-binding protein 2 beta)5094507609tumor necrosis factor (ligand) superfamily, member 95104507643tumor protein D52-like 2; hD545114507645triosephosphate isomerase 15124507645triosephosphate isomerase 15134507665tyrosylprotein sulfotransferase 15144507677tumor rejection antigen (gp96) 1; Tumor rejection antigen-1 (gp96)5154507713tetratricopeptide repeat domain 25164507733Tu translation elongation factor, mitochondrial5174507783ubiquitin-conjugating enzyme E2H (homologous to yeast UBC8)5184507789ubiquitin-conjugating enzyme E2L 35194507793ubiquitin-conjugating enzyme E2N5204507841ubiquinol-cytochrome c reductase core protein I5214507843ubiquinol-cytochrome c reductase core protein II5224507853ubiquitin specific protease, proto-oncogene; Unph5234507857ubiquitin specific protease 7 (herpes virus-associated)5244507879voltage-dependent anion channel 15254507913WAS protein family, member 1; WASP family Verprolin-homologousprotein; scar, dictyostelium, homology of, 15264507953tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein, zeta polypeptide; Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation5274507963zinc finger protein homologous to Zfp37 in mouse5284507979zinc finger protein 1325294522026Bassoon protein; match to PID: g3043642; similar to PID: g3413810, C-terminus matches KIAA0559, N-terminus similar to5304529887NG355314557032lactate dehydrogenase B5324557036microseminoprotein, beta5334557044propionyl Coenzyme A carboxylase, beta polypeptide5344557235acyl-CoA dehydrogenase very long chain5354557247acylphosphatase 2, muscle type5364557265beta-1-adrenergic receptor gi|15298066|ref|XP5374557305aldolase A protein5384557311adenosine monophosphate deaminase 1 (isoform M)5394557317annexin XI5404557365Bloom syndrome protein5414557403carnitine/acylcarnitine translocase; Carnitine-acylcarnitine translocase;carnitine-acylcarnitine carrier; solute carrier family 25(carnitine/acylcarnitine translocase), member 205424557403carrier carnitine-acylcarnitine translocase5434557409cardiac calsequestrin 25444557439cyclin-dependent kinase 35454557451chromodomain helicase DNA binding protein 3; Mi-2a; zinc-fingerhelicase (Snf2-like)5464557565excision repair cross-complementing rodent repair deficiency,complementation group 65474557579fatty acid binding protein 4, adipocyte; A-FABP5484557657immature colon carcinoma transcript 15494557735monoamine oxidase A5504557759myeloperoxidase55145577655-methyltetrahydrofolate-homocysteine methyltransferase; 5-methyltetrahydrofolate-homocysteine methyltransferase 15524557767methylmalonyl Coenzyme A mutase precursor5534557769mevalonate kinase5544557771protein C, cardiac; myosin-binding protein C, cardiac5554557775myosin light chain 25564557817Succinyl CoA: 3-oxoacid CoA transferase5574557817Succinyl CoA: 3-oxoacid CoA transferase; succinyl-CoA: 3-ketoacid-CoAtransferase precursor5584557833Propionyl-Coenzyme A carboxylase, alpha polypeptide5594557845ribonucleotide reductase M2 polypeptide5604557867sulfite oxidase5614557867sulfite oxidase, mitochondrial5624557876ATP-binding cassette, sub-family A member 4; ATP binding cassettetransporter; ATP-binding transporter, retina-specific; rim protein5634587083MRP55644589504Unknown5654589644Unknown5664678807Unknown5674680705CGI-33 protein5684680721thyroid peroxidase5694689104ND ASHI5704730927spermatogenesis associated PD15714757732programmed cell death 8 (apoptosis-inducing factor)5724757762ring finger protein 14; androgen receptor associated protein5734757786N-acylsphingosine amidohydrolase (acid ceramidase)5744757852BCS1 (yeast homolog)-like5754758024coilin; coilin p805764758030coatomer protein complex, subunit alpha; alpha coat protein; xenin5774758038cox 5a5784758040cox 6c5794758118mitochondrial ribosomal protein S29, 28S death associated protein 3;5804758118mitochondrial ribosomal protein S29, 28S death associated protein 3;5814758120death-associated protein 15824758156diacylglycerol kinase, iota5834758192serine/threonine kinase 17a (apoptosis-inducing)5844758242early development regulator 2; homolog of polyhomeotic 25854758312electron-transferring-flavoprotein dehydrogenase5864758352ferredoxin 1 precursor; adrenodoxin5874758490GTP binding protein 15884758498hexose-6-phosphate dehydrogenase precursor5894758504hydroxyacyl-Coenzyme A dehydrogenase, type II5904758520hect domain and RLD 25914758520hect domain and RLD 25924758570heat shock 70 kD protein 9B (mortalin-2); heat shock 70 kD protein 9(mortalin); Heat-shock 70 kD protein-9 (mortalin); mot-2; mthsp755934758582isocitrate dehydrogenase 3 (NAD+) gamma5944758604interleukin enhancer binding factor 3, 90 kD; M-phase phosphoprotein 4;nuclear factor associated with dsRNA5954758664acetylglucosaminyltransferase-like protein5964758682protease, serine, 15; Lon protease-like protein5974758714microsomal glutathione S-transferase 35984758750myosin IXB5994758768ND 42k6004758772ND B96014758774ND 22k, PDSW6024758776ND 7k6034758778ND 8k, AGGG6044758784ND B14.56054758786ND 49k6064758788ND 30k6074758790ND 15k6084758792ND 13 k-A6094758818Notch homolog 4 (Drosophila); Notch, drosophila, homolog of, 4; Notch(Drosophila) homolog 46104758832neuregulin 2 isoform 1; neural- and thymus-derived activator for ErbBkinases6114758852organic cation transporter like 36124758940chromosome 14 open reading frame 2; mitochondrial proteolipid 68 MPhomolog6134758940mitochondrial proteolipid 68 MP homolog6144759020RAB5C, member RAS oncogene family; RAB, member of RAS oncogenefamily-like; RAB5C, member of RAS oncogene family6154759068cytochrome oxidase deficient homolog 16164759080succinate dehydrogenase complex, subunit A, flavoprotein precursor;succinate dehydrogenase complex flavoprotein subunit precursor6174759080succinate dehydrogenase, subunit A, flavoprotein (Fp)6184759082serum deprivation response (phosphatidylserine-binding protein)6194759112solute carrier family 16 (monocarboxylic acid transporters), member 3;monocarboxylate transporter 36204759144carrier family 9 (sodium/hydrogen exchanger), isoform 56214759146slit homolog 2 (Drosophila); slit (Drosophila) homolog 26224759160small nuclear ribonucleoprotein D3 polypeptide6234759196symplekin6244760549IDN36254761539voltage-dependent calcium channel alpha 1G subunit b isoform6264826643annexin A36274826649mitochondrial ribosomal protein L496284826649mitochondrial ribosomal protein L49; chromosome 11 open reading frame 46294826655calbindin 16304826661nuclear receptor subfamily 1, group I, member 36314826661nuclear receptor subfamily 1, group I, member 3; constitutive androstanereceptor-beta; orphan nuclear hormone receptor6324826772insulin-like growth factor binding protein, acid labile subunit6334826848ND B136344826850ND B14.5a NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7(14.5 kD, B14.5a)6354826852ND 8k6364826856ND 75K NADH dehydrogenase (ubiquinone) Fe-S protein 1 (75 kD)(NADH-coenzyme Q reductase); NADH dehydrogenase (ubiquinone), Fe-S protein-1 (75 kD); NADH-ubiquinone oxidoreductase 75 kD subunitprecursor6374826898profilin 16384826914phospholipase A2, group IVB6394826950kallikrein 76404827065zinc finger protein 1476414877291receptor for Advanced Glycation End Products6424885281glutamate dehydrogenase 16434885331G protein-coupled receptor 426444885389hydroxyacyl glutathione hydrolase; glyoxalase 26454885389hydroxyacyl glutathione hydrolase; hydroxyacyl glutathione hydrolase;glyoxalase 2; Hydroxyacyl glutathione hydrolase; glyoxalase II;hydroxyacylglutathione hydroxylase6464885401cytochrome c heme lyase6474885533peptidylglycine alpha-amidating monooxygenase COOH-terminal6484885553postmeiotic segregation increased 2-like 96494885565peroxisomal acyl-CoA thioesterase6504885615signal transducer and activator of transcription 2, 113 kD6514885665achaete-scute complex homolog-like 2; achaete-scute complex(Drosophila) homolog-like 26524887552MUC-B16534894370ND B226544914601Unknown6554929697CGI-114 protein6565031609branched chain alpha-ketoacid dehydrogenase kinase6575031631CD36 antigen6585031691chromosome 21 open reading frame 33; human HES1 protein, homologto E. coli and zebrafish ES1 protein6595031707glycoprotein A repetitions predominant precursor; garpin6605031777isocitrate dehydrogenase 3 (NAD+) alpha6615031777isocitrate dehydrogenase 3 alpha6625031875lamin A/C6635031881leucyl/cystinyl aminopeptidase; leucyl/cystinyl aminopeptidase(oxytocinase)6645031943transcription factor NSCL-1 helix-loop-helix protein6655031987peptidylprolyl isomerase F MITOCHONDRIAL PRECURSOR(6665032017RAD50 (S. cerevisiae) homolog6675032051ribosomal protein S14 40S6685032095carrier family 21 (prostaglandin transporter), member 26695032181translocase of inner mitochondrial membrane Tim17b6705032215translational inhibitor protein6715051381FK506 binding protein 12-rapamycin associated protein 16725059062pilin-like transcription factor6735114261voltage-dependent anion channel isoform 26745138999NADH-Ubiquinone reductase6755174539malate dehydrogenase 1, NAD (soluble)6765174539malate dehydrogenase 1, NAD (soluble); Malate dehydrogenase, soluble6775174541malate dehydrogenase 2, NAD (mitochondrial); Malate dehydrogenase,mitochondrial6785174563MHC binding factor, beta6795174627plasma glutamate carboxypeptidase; aminopeptidase6805174739tubulin, beta, 56815174743ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 16825360087NY-REN-6 antigen6835453549thioredoxin peroxidase; thioredoxin peroxidase (antioxidant enzyme)6845453559ATPase d F06855453670golgi transport complex 1 (90 kD subunit); golgi transport complex 1 (90 kDasubunit)6865453750brain acid-soluble protein 1; neuronal tissue-enriched acidic protein6875453890PIBF1 gene product6885453902NIMA-interacting, 4 (parvulin) peptidyl-prolyl cis-trans isomerase EPVH6895453990proteasome (prosome, macropain) activator subunit 1 (PA28 alpha)6905454028related RAS viral (r-ras) oncogene homolog; Oncogene RRAS6915454122translocase of inner mitochondrial membrane Tim236925454148UNC136935454152ubiquinol-cytochrome c reductase binding protein6945454180zinc finger protein 1936955578989Unknown6965689405Unknown6975689555Unknown6985701717UDP-N-acetylglucosamine:alpha-1,3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV-homologue6995725250G7 protein7005725370involved in chromosomal translocation7015729802Unknown7025729875progesterone binding protein7035729877heat shock 70 kD protein 8; heat shock 70 kD protein 8 (HSP73); heatshock cognate protein, 71-kDa; heat shock 70 kd protein 10 (HSC71)7045729887IQ motif containing GTPase activating protein 2, RasGAP-related protein7055729937metaxin 27065729937metaxin 27075729966MHC class I region ORF7085730027GAP-associated tyrosine phosphoprotein p62 (Sam68)7095730033sodium channel, voltage-gated, type X, alpha polypeptide7105730110ubiquitin specific protease 3 gi|10720340|sp|Q9Y6I4|UBP3_HUMANUBIQUITIN CARBOXYL-TERMINAL HYDROLASE 37115759173succinate dehydrogenase flavoprotein subunit7125802182PPAR gamma coactivator-17135802814Gag-Pro-Pol-Env protein7145802970AFG3 (ATPase family gene 3, yeast)-like 27155803115mitofilin inner membrane protein, mitochondrial (mitofilin); motor protein7165803135RAB35, member RAS oncogene family; ras-related protein rab-17175803149coated vesicle membrane protein7185803159sex comb on midleg (Drosophila)-like 17195803201transmembrane trafficking protein7205803207U2 small nuclear RNA auxillary factor 1; U2 snRNP auxiliary factor smallsubunit; splicing factor U2AF 35 kDa subunit7215821952Rotamer Strain As A Determinant Of Protein Structural Specificity7225882259genethonin 37235901896ATPase epsilon F17245901926cleavage and polyadenylation specific factor 5, 25 kD subunit7255901982isocitrate dehydrogenase 3 (NAD+) beta7265902106SRY (sex determining region Y)-box 207275902110SRY (sex determining region Y)-box 22; SPY (sex-determining region Y)-box 227285924409tight junction protein ZO-2 isoform C7296005717ATPase e F07306005772putative G protein coupled receptor7316005938utrophin; dystrophin-related protein7326005938utrophin; dystrophin-related protein7336005948WW domain-containing binding protein 4; formin binding protein 217346010711hereditary haemochromatosis protein precursor7356031192phosphate carrier precursor isoform 1a; phosphate carrier, mitochondrial;phosphate carrier, mitochondrial precursor7366041669ND B157376094658truncated form of cytochrome Bc1 J chain; similar to 1BGY7386175038Son of sevenless protein homolog 2 (SOS-2)7396176530alanine-glyoxylate aminotransferase homolog7406249687R31155_17416273778trabeculin-alpha7426274550ND B22 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9 (22 kD,B22)7436288790beta-ureidopropionase7446330385Unknown7456331429Unknown7466382058v-abl Abelson murine leukemia viral oncogene homolog 1 isoform b;Abelson murine leukemia viral (v-abl) oncogene homolog 17476382071diaphanous 2 isoform 12C; Diaphanous, Drosophila, homolog of, 2;diaphanous (Drosophila, homolog) 27486433936aczonin7496456828phosphoglycerate kinase 17506523797adrenal gland protein AD-0027516572219UCR ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide-like 1) dJ370M22.2 (7526580492cN28H9.1 (novel protein)7536594629pRGR27546598323GDP dissociation inhibitor 2; rab GDP-dissociation inhibitor, beta75566241223-hydroxyisobutyrate dehydrogenase7566631100natural killer-tumor recognition sequence7576649914growth/differentiation factor-117586678455transcription termination factor, RNA polymerase I7596681764ND 39k NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9(39 kD); NADH dehydrogenase (ubiquinone) Fe-S protein 2-like (NADH-coenzyme Q reductase)7606683124Unknown7616686262ZINC FINGER PROTEIN 367626688130poly-(ADP-ribose) polymerase II7636729803Heat-Shock 70 kd Protein 42 kd Atpase N-Terminal Domain7646739500LDLR-FUT fusion protein7656841066calcium-binding transporter7666841110Unknown7676841194HSPC2727686841440HSPC1087696841930T cell receptor beta chain7706912238peroxiredoxin 5; antioxidant enzyme B1667716912322crumbs homolog 1; crumbs (Drosophila) homolog 17726912396glyoxylate reductase/hydroxypyruvate reductase7736912440double-stranded RNA-binding zinc finger protein JAZ7746912482LETM1 leucine zipper-EF-hand containing transmembrane protein 17756912482leucine zipper-EF-hand containing transmembrane protein 17766912536nicotinamide nucleotide transhydrogenase7776912536nicotinamide nucleotide transhydrogenase7786912538neurotensin receptor 2; neurotensin receptor, type 27796912664sirtuin 5, isoform 1; sir2-like 5; sirtuin type 5; sirtuin (silent mating typeinformation regulation 2, S. cerevisiae, homolog) 5; silent mating typeinformation regulation 2, S. cerevisiae, homolog 57806912714translocase of inner mitochondrial membrane 9 homolog (yeast);translocase of inner mitochondrial membrane 9 (yeast) homolog7816912714translocase of inner mitochondrial membrane Tim9a7826996429acetyl-coenzyme A synthethase (acetate-coA ligase)) dJ568C11.3 (novelAMP-binding enzyme similar to7836996429novel AMP-binding enzyme similar to acetyl-coenzyme A synthethase(acetate-coA ligase)7847018398hemopoietic cell kinase7857019351cardiotrophin-like cytokine; neurotrophin-1/B-cell stimulating factor-37867019545secreted protein of unknown function7877020216Unknown7887020807mitochondrial ribosomal protein L22, similar to7897022241Unknown7907022343Unknown7917022728Unknown7927022751Unknown7937242949Unknown7947242979Unknown7957243141Unknown7967243219Unknown7977243272Unknown7987243280Unknown7997245352Hexokinase I With Glucose And Adp In The Active Site, Mutant MonomerOf Recombinant Human8007329718Unknown8017430427ionizing radiation resistance conferring protein - human8027431153malate dehydrogenase (EC 1.1.1.37), cytosolic - human8037431833NAD(P)+ transhydrogenase (B-specific) (EC 1.6.1.1) precursor,mitochondrial - human8047436377plasma membrane Ca2+-ATPase variant 4a PMCA4a - human(fragment)8057439346protein-tyrosine-phosphatase8067441369tubulin beta chain - human8077447071syntaxin8087447698UDP glucuronosyltransferase (EC 2.4.1.—) 1A10 precursor - human8097452946X-like 1 protein8107459551Unknown8117487801Unknown8127511895Unknown8137512435filamin, muscle8147512482helicase II - human8157512482helicase II - human gi|606833|gb|AAC50069.1|(U09820) helicase II8167512513Unknown8177512598Unknown8187512628Unknown8197512754Unknown8207512754Unknown8217512776Unknown8227512977Unknown8237513005Unknown8247513021Unknown8257513022Unknown8267513076Unknown8277513172N-chimerin homolog F25965_3 - human8287513177ND 14.1K NADH dehydrogenase (ubiquinone) (EC 1.6.5.3) 14.1K chain -human8297513178ND acyl carrier NADH dehydrogenase (ubiquinone) (EC 1.6.5.3) acylcarrier chain, mitochondrial - human (fragment)8307513274probable thyroid receptor interactor - human (fragment)8317513374thrombospondin-p50 - human (fragment)8327524346adenylate kinase 2 isoform b; Adenylate kinase-2, mitochondrial8337527760Unknown8347582306ALEX3 protein8357595299opioid growth factor receptor8367643782HDCMD47P8377656959calpain 7; calpain like protease;8387656999catenin8397657039death receptor 68407657050hypothetical protein, estradiol-induced8417657257translocase of outer mitochondrial membrane 20 (yeast) homolog8427657257translocase of outer mitochondrial membrane 20homolog (TOM20)8437657343metalloprotease 1 (pitrilysin family)8447657347mitochondrial carrier homolog 28457657347mitochondrial carrier homolog 28467657369ND 19k NDUFA88477657469rat integral membrane glycoprotein POM121, similar to8487657486low molecular mass ubiquinone-binding protein8497657534spastic ataxia of Charlevoix-Saguenay8507657554soggy-1 gene; dickkopf-like 1 (soggy)8517657562SH3-domain binding protein 48527657581solute carrier family 25, member 13 (citrin)8537657615podocin8547661602DKFZP564B167 protein8557661602Unknown8567661678RAS-related protein RAP1B; K-REV DKFZP586H0723 protein;8577661720HIRA interacting protein 5; HIRIP5 protein; HIRA-interacting protein 5;HIRA-interacting protein 58587661732HSPC009 protein8597661732Unknown8607661800HSPC141 protein8617661872leucyl-tRNA synthetase, mitochondrial8627661872leucyl-tRNA synthetase, mitochondrial; KIAA0028 protein8637661960Rough Deal homolog, centromere/kinetochore protein; Rough Deal(Drosophila) homolog, centromere/kinetochore protein8647661996Unknown8657662042Rho guanine nucleotide exchange factor 108667662046Unknown8677662092Unknown8687662168Unknown8697662190Unknown8707662190Unknown8717662280histone deacetylase 7B isoform HDRP; histone deacetylase 7; MEF-2interacting transcription repressor (MITR) protein; histone deacetylase 7B8727662284Unknown8737662314Unknown8747662452Unknown8757662470neuroligin 18767662480Unknown8777662639PTD011 protein8787662645mitochondrial ribosomal protein S18B; mitochondrial ribosomal proteinS18-2; mitochondrial 28S ribosomal protein S18-28797662673translocase of outer mitochondrial membrane 70 homolog A (yeast);translocase of outer mitochondrial membrane 70 (yeast) homolog A;KIAA0719 gene product8807662673translocase of outer mitochondrial membrane 70homolog A8817669477RNA-specific adenosine deaminase B1, isoform DRABA2b; RNA editase;human dsRNA adenosine deaminase DRADA2b8827669492glyceraldehyde-3-phosphate dehydrogenase8837669520neuregulin 1 isoform ndf43; heregulin, alpha (45 kD, ERBB2 p 185-activator); glial growth factor8847671629KRAB box containing C2H2 type zinc finger protein8857671653Unknown8867677070silent information regulator 2 homolog8877678804mitochondrial isoleucine tRNA synthetase8887705485Unknown8897705501Unknown8907705594CGI-10 protein8917705616CGI-112 protein8927705626mitochondrial ribosomal protein S168937705626mitochondrial ribosomal protein S16; 28S ribosomal protein S16,mitochondrial8947705646CGI-150 protein8957705704glutathione S-transferase subunit 13 homolog mitochondrial8967705738mitochondrial ribosomal protein S7; 30S ribosomal protein S7 homolog8977705797CGI-87 protein8987705805mitochondrial ribosomal protein S28997705805mitochondrial ribosomal protein S29007705889NEU1 protein9017705987glycolipid transfer protein9027706057mitochondrial ribosomal protein L279037706073GS159047706117peptide transporter 3; likely ortholog of rat peptide/histidine transporter 29057706121testicular haploid expressed gene9067706146hBOIT for potent brain type organic ion transporter9077706154NM23-H89087706314CGI-77 protein9097706349mitochondrial ribosomal protein S339107706449fatty-acid-Coenzyme A ligase, long-chain 5; long-chain acyl-CoAsynthetase 5; long-chain fatty acid coenzyme A ligase 5; FACL5 for fattyacid coenzyme A ligase 59117706481MO25 protein9127706549CDC2-related protein kinase 79137710129LIM domain only 69147770231Unknown9157799988large-conductance calcium-activated potassium channel beta9167959706Unknown9177959889Unknown9187959907PRO24729197981263Unknown9208051579adenylate kinase 3; Adenylate kinase-3, mitochondrial; GTP: AMPphosphotransferase9218131894mitofilin9228216989putative cell cycle control protein9238217423bA108L7.7 (novel protein similar to C. elegans C25A1.13 (Tr: O02220))9248394499ubiquitin associated protein9258488995ND 20K NADH-ubiquinone oxidoreductase 20 kDa subunit, mitochondrialprecursor (Complex I-20 KD) (CI-20 KD) (PSST subunit)9268570444Contains similarity to an unnamed protein from Homo sapiens9278574030diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme Abinding protein))) dJ1013A10.3 (related to DBI (9288574070NFKB19298671846RNA adenosine deaminase gene, exon 15, Contains similarity to9308919645T-cell receptor beta chain9318922081Unknown9328922081Unknown9338922275Unknown9348922285Unknown9358922307Unknown9368922420neuropilin and tolloid like-29378922465Unknown9388922511mitochondrial ribosomal protein S18A9398922517Unknown9408922569Unknown9418922629Unknown9428922665Unknown9438922701putative lipid kinase9448922742Unknown9458922787Unknown9468922804Unknown9478922838Unknown9488923001Unknown9498923221Unknown9508923291Unknown9518923390Unknown9528923390Unknown9538923415Unknown9548923417Unknown9558923528Unknown9568923870hOAT49578923930uncharacterized hematopoietic stem/progenitor cells protein9588923930uncharacterized hematopoietic stem/progenitor cells protein MDS09598927581testes-specific heterogenous nuclear ribonucleoprotein G-T9608928067Malonyl-CoA decarboxylase, mitochondrial precursor (MCD)96190493523-methylcrotonyl-CoA carboxylase biotin-containing subunit9629256610protocadherin beta 15 precursor9639257242succinate dehydrogenase complex, subunit B, iron sulfur (Ip); iron-sulfursubunit9649296943Cyclin T29659297078UBIQUINOL-CYTOCHROME C REDUCTASE COMPLEX 7.2 KDAPROTEIN9669367862Unknown9679438229phospholipase C beta 19689501146meiotic DNA transesterase/topoisomerase homolog isoform 29699506437FAPP1-associated protein 19709506611Unknown9719506611Unknown9729506637rab11-binding protein gi|7023581|dbj|BAA92015.1|(AK001978) unnamedprotein product, similar to9739506697Unknown9749506713nucleolar protein family A, member 1; H/ACA small nucleolar RNPsprotein 19759506785homeo box (H6 family) 19769622528NSAID-activated protein 1 NAG-19779884738AP-2 beta transcription factor9789910184DC13 protein9799910244mitochondrial ribosomal protein S22; gibt protein; chromosome 3 openreading frame 5; mitochondrial 28S ribosomal protein S229809910280UDP-glucose ceramide glucosyltransferase-like 19819910382mitochondrial import receptor Tom229829910382mitochondrial import receptor Tom229839911130protein phosphatase9849930803A kinase (PRKA) anchor protein 79859955433Unknown9869966799disrupter of silencing 109879966893CGI-203 protein98810047106carboxypeptidase A398910047118G-protein gamma-12 subunit99010047120insulin receptor tyrosine kinase substrate99110047167Unknown99210047177Unknown99310047183Unknown99410047187Unknown99510047199Unknown99610047213Unknown99710047231Unknown99810047239Unknown99910047243Unknown100010047247Unknown100110047249Unknown100210047277Sarcolemmal-associated protein100310047277Unknown100410047279Unknown100510047281Unknown100610047283Unknown100710047317L-periaxin100810047329Unknown100910047335zinc finger protein101010047341Unknown101110047341Unknown101210047347Unknown101310047361Unknown101410092604HUG1 gene101510092623hematopoietic PBX-interacting protein gi|993010161009265713 kDa differentiation-associated protein; NADH: ubiquinoneoxidoreductase101710092657ND B17.2101810120604L-3-Hydroxyacyl-Coa Dehydrogenase Complexed With Acetoacetyl-CoaAnd Nad+101910179599ND NDUFS2102010179880muscle-specific protein102110181206GABA(A) receptor-associated protein like 1102210190653sphingosine-1-phosphate lyase 1102310190692junctophilin 3; junctophilin type3 gi|9886738102410241702putative ZIC3 Binding protein from Xenopus laevis, similar to102510241706Unknown102610257409natural resistance-associated macrophage protein 1102710257494N-ethylmaleimide-sensitive factor102810334442hydroxysteroid (17-beta) dehydrogenase 7102910334443Unknown103010334466Unknown103110337605peroxisomal short-chain alcohol dehydrogenase103210432782testin103310432971Unknown103410433147poly(A) polymerase gamma; SRP RNA 3′ adenylating enzyme/pap2103510433320huntingtin-associated protein103610433905Unknown103710433929Unknown103810434023Unknown103910434055Unknown104010434106Fanconi anemia complementation group D2 protein104110434151Unknown104210434167Unknown104310434183Unknown104410434243Unknown104510434293Unknown104610434345Unknown104710434521Unknown104810434757Unknown104910434850zinc finger protein 226105010434904Unknown105110434988Unknown105210435007Unknown105310435244Unknown105410435551Unknown105510435767Unknown105610435899Unknown105710435947Unknown105810436007Unknown105910436258Unknown106010436263Unknown106110436325Unknown106210436604Unknown106310437144Smac106410437144Unknown106510437178mitochondrial ribosomal protein L1106610437189Unknown106710437384M-phase phosphoprotein 1106810437960Unknown106910437984Unknown107010438291Unknown107110438353McKusick-Kaufman syndrome protein107210438441Unknown107310438702Unknown107410438857Unknown107510438928mitochondrial ribosomal protein S11107610438968Unknown107710439079Unknown107810439244Unknown107910439312Unknown108010440252bromodomain PHD finger transcription factor108110440347Unknown108210440357Unknown108310440367Unknown108410440389Unknown108510440402Unknown108610440484Unknown108710441879Unknown108810441930Unknown108910443472Rhesus blood group-associated glycoprotein (RH50A)109010503988Unknown109110518340muscleblind (Drosophila)-like109210567164gene amplified in squamous cell carcinoma-1109310639097solute carrier family 24 (sodium/potassium/calcium exchanger), member3) dJ122P22.1 (109410645199ADAM-TS disintegrin and metalloprotease with thrombospondin motifs-7preproprotein; a disintegrin-like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 7109510716563calnexin109610719935CELL DIVISION CYCLE 2-LIKE PROTEIN KINASE 5(CDC2-RELATED PROTEINKINASE 5)109710720290SORTING NEXIN 14109810720297SYNAPTOJANIN 2 (SYNAPTIC INOSITOL-1,4,5-TRISPHOSPHATE 5-PHOSPHATASE 2)109910720409Zinc finger protein 294110010764847ND B18110110798812MLTK-alpha110210834587fer-1 like protein 3110310834762PNAS-102110410834786PNAS-117110510834968mannosidase, alpha B, lysosomal110610835000pancreatic lipase110710835002Rho GDP dissociation inhibitor (GDI) beta110810835023inositol 1,4,5-triphosphate receptor, type 1110910835025ND 24k111010835045retinaldehyde dehydrogenase 2111110835057N-acetyltransferase, homolog of S. cerevisiae ARD1; N-acetyltransferaseARD1, human homolog of111210835059farnesyltransferase, CAAX box, beta111310835063nucleophosmin (nucleolar phosphoprotein B23, numatrin)111410835087ND 10k111510835089neurofilament, heavy polypeptide (200 kD); Neurofilament, heavypolypeptide111610835109myotubularin related protein 3; FYVE (Fab1 YGLO23 Vsp27 EEA1domain) dual-specificity protein phosphatase111710835155tumor necrosis factor (cachectin)111810835165CD59 antigen p18-20111910835173nitric oxide synthase 1112010835189glutathione reductase112110835220ATPase, Ca++ transporting, fast twitch 1112210863907hepatocellular carcinoma associated protein; breast cancer112310863927peptidylprolyl isomerase A112410863945ATP-dependant DNA helicase II112510863985G4 protein112610864011CGI-44 protein; sulfide dehydrogenase like (yeast)112710864043kidney and liver proline oxidase 1112810864077calcium channel, voltage-dependent, alpha 1H subunit112910945428membrane-associated guanylate kinase MAGI3113011024710Unknown113111024714ubiquitin B113211034855Unknown113311038674CD79B antigen, isoform 1 precursor; B-cell-specific glycoprotein B29113411055998guanine nucleotide binding protein beta subunit 4 [Homo sapi113511056030protocadherin gamma subfamily A, 2, isoform 1 precursor113611066958mutant beta-globin113711066968EH domain-containing protein FKSG7113811095436valosin-containing protein113911096171RNA polymerase III transcription initiation factor B114011121497Trp4-associated protein TAP1, similar to114111127695SYT/SSX4 fusion protein114211128019cytochrome c114311128031protocadherin gamma subfamily B, 5, isoform 1 precursor114411139093GrpE-like protein cochaperone114511141885carrier family 5 (choline transporter), member 7114611141891ERGL protein114711177148mitochondrial ribosomal protein L12114811177148mitoribosomal protein L12114911225260DNA TOPOISOMERASE I115011225266transient receptor potential cation channel, subfamily M, member 5;MLSN1- and TRP-related; MLSN1 and TRP-related115111245229ninein-Lm isoform115211252721glutaryl-CoA dehydrogenase115311252721glutaryl-CoA dehydrogenase (EC 1.3.99.7) [imported] - human115411267525probable RNA helicase115511275568mucin 5B115611275986glycerol-3-phosphate dehydrogenase 3115711276083fatty-acid-Coenzyme A ligase, long-chain 2115811276083long-chain fatty-acid-Coenzyme A ligase 2; acyl-activating enzyme; acyl-CoA synthetase; fatty acid thiokinase (long-chain); lignoceroyl-CoAsynthase; long-chain acyl-CoA synthetase 2115911276655ribosomal protein S26 [imported] - human116011276938villin 2116111277141heat shock 90 kD protein beta116211280538Unknown116311280677Unknown116411281511Unknown116511321341MondoA116611321569olfactory receptor, family 3, subfamily A, member 2116711321571slit homolog 3 (Drosophila); slit (Drosophila) homolog 3; slit (Drosophila)homolog 2; slit2116811321579myosin, heavy polypeptide 13, skeletal muscle; extraocular musclemyosin heavy chain116911321581succinyl-CoA synthetase alpha subunit117011321583succinate-CoA ligase, ADP-forming, beta subunit117111321613epilepsy, progressive myoclonus type 2, Lafora disease (laforin)117211321615T-box 3 protein; T-box 3; T-box transcription factor TBX3117311323320ubiquitin-conjugating enzyme E2 variant 1 (isoform 2, similar to variant 2(UBE2V2, MMS2)117411342570metalloproteinase 24 (membrane-inserted), matrix117511342672myosin, heavy polypeptide 3, skeletal muscle, embryonic117611345448lipopolysaccharide-binding protein117711345456fibroblast growth factor receptor-like 1 precursor117811345478Unknown117911345539novel Helicase C-terminal domain118011359874GTP-binding protein 2118111359883Unknown118211359946leucine zipper-EF-hand containing transmembrane protein 1118311359985Unknown118411359986Unknown118511360009Bcl-Rambo118611360009Unknown118711360063matrilin 2 precursor118811360067Unknown118911360079Unknown119011360112Unknown119111360155Unknown119211360155Unknown119311360156Unknown119411360162Unknown119511360185Unknown119611360188Unknown119711360228Unknown119811360250Unknown119911360251Unknown120011360294Unknown120111360310myosin Vlla, long form - human120211360321properdin120311374664isocitrate dehydrogenase (EC 1.1.1.42), cytosolic120411385354polybromo 1120511385644CTCL tumor antigen se2-1120611385664CTCL tumor antigen se89-1120711386147prosaposin120811399466D-2-hydroxy-acid dehydrogenase120911415024diacylglycerol kinase, alpha (80 kD)121011416393mitochondrial ribosomal protein L22121111416669nicotinamide nucleotide transhydrogenase121211417363low molecular mass ubiquinone-binding protein121311417363low molecular mass ubiquinone-binding protein121411418549eyes absent (Drosophila) homolog 4121511418714Unknown121611419832phosphorylase kinase, alpha 1121711421027Unknown121811422272ribosomal protein S6 kinase, 90 kD121911423142basic leucine zipper nuclear factor 1122011423880alpha-SNAP122111424404mitochondrial ribosomal protein S23122211424724neurofilament 3122311425565Unknown122411425836low density lipoprotein receptor-related protein 3122511427613Unknown122611427636GTPase Rab14122711428230aldehyde dehydrogenase 1 family, member B1122811429803Unknown122911430299hexokinase 1123011431667multiple inositol polyphosphate phosphatase 2123111432018Unknown123211432441Unknown123311432489general transcription factor IIE, polypeptide 1 (alpha subunit, 56 kD)123411433007peroxisomal enoyl-coenzyme A hydratase-like protein123511433596tryptophanyl-tRNA synthetase123611434079Unknown123711434447Unknown123811434986COQ6_HUMAN PUTATIVE UBIQUINONE BIOSYNTHESISMONOOXGENASE COQ123911435257Unknown124011435724mannosidase, beta A, lysosomal124111436135RAS-RELATED PROTEIN R-RAS2124211436533aldehyde dehydrogenase 2 family (mitochondrial)124311436778inositol polyphosphate-4-phosphatase, type II, 105 kD124411437205Unknown124511440003transgelin124611441230skeletal muscle specific actinin, alpha 3124711493459PRO2619124811493489PRO2620124911493522Unknown125011493552Unknown125111496882ELK4 protein, isoform b; ETS-domain protein; SRF accessory protein 1125211497601metallaproteinase-disintegrin125311526149ATPase CF6 F0125411526456frataxin125511526471tripartite motif protein TRIM14 isoform alpha125611526573heat shock cognate protein 54125711526789inorganic pyrophosphatase 2125811545761potassium channel, subfamily K, member 12; tandem pore domainpotassium channel THIK-2125911545847basic-helix-loop-helix-PAS protein126011545863methylcrotonoyl-Coenzyme A carboxylase 2126111545869popeye protein 2126211545894RFamide-related peptide precursor126311559927mitochondrial ribosomal protein S14126411596402MAGE-D4126511596859mitochondrial ribosomal protein L17126611602741complement component 8, alpha polypeptide126711602963heparan sulfate proteoglycan perlecan126811611734GREB1a126911612659FXYD domain-containing ion transport regulator 7127011612670phospholemman, isoform b precursor; FXYD domain-containing127111640566hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme Athiolase/enoyl-Coenzyme A hydratase beta127211640578glyoxylate reductase/hydroxypyruvate reductase127311641249protein kinase Njmu-R1127411641283LIM homeobox protein 5127511641413cell division cycle 25B, isoform 3; CDC25B127611761696bHLHZip transcription factor BIGMAX gamma127711863673guanine nucleotide binding protein (G protein), alpha stimulating activitypolypeptide 1) dJ309F20.1.5 (isoform 5 of127811890755RNA helicase II/Gu protein127911907570mutant desmin128011908171Fas-binding protein Daxx128111935053sarcolemmal associated protein 11282119680035-azacytidine induced gene 2, similar to128311968152somatostatin receptor-interacting protein128411990879phosphoglycerate kinase 2128511991867odorant receptor HOR3′beta5128612001946My003 protein128712001986My022 protein128812001992brain my025128912002038brain my045 protein129012002042brain my048 protein129112002201serine/threonine protein kinase PFTAIRE-1129212003293organic anion transporter 2129312005493NPD011129412005918GRIM19129512006049EF1a-like protein129612006205TNFIP-iso129712038977Unknown129812043738thioredoxin reductase, mitochondrial129912052810Unknown130012052820COQ7 protein; timing protein; ubiquinone biosynthesis protein130112052826RAB-8b protein, small GTP-binding protein130212052828Unknown130312052872Unknown130412052908Unknown130512052971methyltransferase COQ3130612052989Unknown130712052991Unknown130812053107Unknown130912053245Unknown131012053255Unknown131112060822serologically defined breast cancer antigen NY-BR-16131212060832serologically defined breast cancer antigen NY-BR-40131312061185ASC-1 complex subunit P200131412081909semaphorin Y131512214171putative small GTP-binding protein (rab1b)131612214288dJ402H5.2 (novel protein similar to worm and fly proteins)131712230015CYTOCHROME B5 OUTER MITOCHONDRIAL MEMBRANE ISOFORMPRECURSOR131812230075GLYCEROL KINASE, TESTIS SPECIFIC 1131912232373rab6 GTPase activating protein (GAP and centrosome-associated)132012232421tricarboxylate carrier protein132112232477Unknown132212239360LYST-interacting protein LIP6132312246901tumor protein D52-like 2132412248755mono ATP-binding cassette protein132512314005Unknown132612314016transcription factor TFIIS, similar to132712314029proteasome subunit 7132812314062Unknown132912314123uncharacterized hematopoietic stem/progenitor cells protein MDS030(8923932)133012314190dJ445H2.2 (novel protein)133112314195Unknown133212328445NPAS3133312382773caspase recruitment domain protein 11133412382789OSBP-related protein 7; ORP7133512383092Unknown133612407403tripartite motif protein TRIM9 isoform alpha133712408656calpain 1, large subunit133812597655kinetochore protein133912620194Unknown134012620246CD36134112620252CD36134212620871phosphoinositide-3-kinase gamma catalytic subunit134312621903cathepsin S134412643256pilin-like transcription factor134512643326CIP1-INTERACTING ZINC FINGER PROTEIN (NUCLEAR PROTEINNP94)134612643329CGI-51134712643417Pyruvate dehydrogenase protein X component, mitochondrial precursor(Dihydrolipoamide dehydrogenase-binding protein of pyruvatedehydrogenase complex) (E3-binding protein) (E3BP) (proX)134812643637ADAM-TS 4 PRECURSOR (A DISINTEGRIN ANDMETALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS 4)134912643716PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 13135012643796RETINOBLASTOMA-BINDING PROTEIN 8135112643896Zinc finger protein 236135212644018AF-6 PROTEIN135312644090T-BOX TRANSCRIPTION FACTOR TBX18135412644310COATOMER BETA SUBUNIT(BETA-COP)135512644370Zinc finger X-linked protein ZXDB135612652715nucleolar GTPase135712652761Unknown135812652763Unknown135912652773Unknown136012652981glycogen synthase kinase 3 beta136112652989Unknown136212653017LRP16 protein136312653371phosphoglycerate mutase 1136412653507aspartate transaminase 2136512653549mitochondrial ribosomal protein S6136612653687Unknown136712653775helicase-like protein NHL136812653827mitochondrial carrier homolog 1 or presenilin-associated protein136912653855dynamitin137012654077NICE-5 protein137112654149Unknown137212654285peptide N-glycanase homolog137312654289transcription termination factor, mitochondrial137412654333HCDI protein137512654407N-Acetylglucosamine kinase137612654521Unknown137712654627metalloprotease 1137812654675transcobalamin II; macrocytic anemia137912655133CGI-63 protein, similar to138012655157centrosomal protein 2138112655195heat shock 75 protein138212656979antigen, T-cell receptor138312657106Unknown138412659007protein kinase D2138512669909long-chain fatty-acid-Coenzyme A ligase 4, isoform 2; long-chain acyl-CoA synthetase 4; acyl-activating enzyme138612697312putative chromatin modulator138712697482novel zinc finger protein similar to rat RIN ZF)138812697776polyadenylation specificity factor138912697899Unknown139012697903Unknown139112697947Unknown139212697951Unknown139312697957Unknown139412697983Unknown139512697991Unknown139612697995Unknown139712698037Unknown139812698043Unknown139912698057likley ortholog of rat CPG2 protein140012698069Unknown140112698075Unknown140212700223recombination activating protein 1140312707570enoyl Coenzyme A hydratase, short chain, 1, mitochondrial140412711660protein kinase, lysine deficient 1140512711664Unknown140612711674yeast Upf3, variant B, similar to140712725813poly(ADP-ribosyl)transferase140812729652cell adhesion molecule with homology to L1CAM (close homologue of L1)140912733033caldesmon 1 or) NAG22 protein141012733091replication initiation region protein (60 kD)141112734392annexin A13141212734816PRP4/STK/WD splicing factor141312735217surfeit 5141412735226adenylate kinase 3 alpha141512735430PKCq-interacting protein PICOT141612738042klotho141712738974Unknown141812740808A kinase anchor protein 10141912741202UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase142012741866protein expressed in thyroid, similar to142112742008chondroitin sulfate proteoglycan 3142212742415complement component C1q receptor142312751117PNAS-140142412751119PNAS-141142512751452PDZ domain-containing protein AIPC142612803243Unknown142712803281VDAC-3142812803349transcription factor 19, similar to142912803387antiquitin 1143012803567transgelin 2143112803843protein kinase, cAMP-dependent, regulatory, type II, alpha, similar to143212803855metastasis suppressor protein143312803915glucosidase I, similar to143412804041nuclear protein E3-3 orf1143512804069FK506-binding protein 4 (59 kD), similar to143612804185colon cancer-associated protein Mic1, similar to143712804225Unknown143812804313expressed sequence 2 embryonic lethal, similar to143912804319carbonyl reductase144012804667Unknown144112804743Unknown144212804755NPD002 protein, similar to144312804821Unknown144412804897branched chain aminotransferase 2, mitochondrial, similar to144512804901isocitrate dehydrogenase 3 gamma144612805021acyl-Coenzyme A dehydrogenase family, member 8144712805031roundabouth144812830367serine/threonine kinase 33144912862320WDC146145012963353fenestrated-endothelial linked structure protein145113027604mitochondrial ribosomal protein S34145213027608Unknown145313027640lysine-ketoglutarate reductase/saccharopine dehydrogenase145413095054ovarian immunoreactive antigen145513096727Smac Bound To Xiap-Bir3 Domain145613096755Ras G12v - Pi 3-Kinase Gamma Complex145713097156ND 39 k145813097243Unknown145913097693Unknown146013111705Carnitine O-acetyltransferase (Carnitine acetylase) (CAT)146113111762solute carrier family 19 (folate transporter), member 1, similar to146213112023coenzyme Q, 7homolog146313123976ARGININE-TRNA-PROTEIN TRANSFERASE 1146413124237F-box only protein 10146513124883HsKin17 protein146613128992Unknown146713128998Unknown146813129014Unknown146913129080Unknown147013129092Unknown147113129144Unknown147213161081testis protein147313177634surfactant protein B-binding protein147413177648EGF factor 8 protein147513177700Unknown147613184052butyrophilin, subfamily 2, member A3147713194197kinesin family member 13B; guanylate kinase associated kinesin147813194522PMF-1 binding protein147913236495quinone oxidoreductase; NADPH148013236559Unknown148113242069nuclear transcription factor NFX2148213242172potassium voltage-gated channel, Shab-related subfamily, member 2148313242739myelin P2 protein148413249985Lowe oculocerebrorenal syndrome protein148513259127cullin CUL4B148613259497retinoblastoma-binding protein 1, isoform I148713272567ND 5148813272568ND 6148913272595ND 5 NADH dehydrogenase subunit 5149013272697ND 1 NADH dehydrogenase subunit 1149113272855ATPase 8149213273190cox 2149313274124Unknown149413276227chromogranin B(isoform 2)149513276598Unknown149613276617Unknown149713278690Unknown149813324710interleukin 3 receptor, alpha (low affinity); Interleukin-3149913325066cadherin EGF LAG seven-pass G-type receptor 3; EGF-like-domain150013325162Unknown150113325394phosphatidylserine synthase 1, similar to150213359201Unknown150313375614peroxisomal long-chain acyl-coA thioesterase150413375634human immunodeficiency virus type I enhancer-binding protein150513375744Unknown150613375809Unknown150713375817Unknown150813375838Unknown150913375872Unknown151013375932Unknown151113375940Unknown151213375942Unknown151313376007Unknown151413376011engulfment and cell motility 3; ced-12 homolog 3151513376021Unknown151613376038Unknown151713376052Unknown151813376093Unknown151913376107Unknown152013376144Unknown152113376284Unknown152213376331Unknown152313376437Unknown152413376445Unknown152513376490Unknown152613376580Unknown152713376617Unknown152813376640putative N-acetyltransferase152913376662Unknown153013376717Unknown153113376741Unknown153213376747Unknown153313376749Unknown153413376776Unknown153513376812type 1 protein phosphatase inhibitor153613376826UL16-binding protein 1153713376854UBX domain-containing 1; UBX domain-containing 2153813376991voltage-dependent calcium channel beta 2 subunit153913386494Unknown154013399777Macrophage Migration Inhibitory Factor (Mif) Complexed With Inhibitor.154113431759PARAPLEGIN154213431763Pre-mRNA cleavage complex II protein Pcf11154313435131WW domain-containing binding protein 4154413435350ferredoxin reductase isoform 1154513436080cleft lip and palate associated transmembrane protein 1154613436188mitochondrial ribosomal protein S2154713436197Unknown154813436275LON PROTEASE HOMOLOG, MITOCHONDRIAL PRECURSOR154913436296Unknown155013436308Unknown155113436335IF-1 ATPase inhibitor precursor155213436395Unknown155313436413glucose phosphate isomerase155413445577EDAG155513449263Unknown155613449269Unknown155713469731breast cancer antigen NY-BR-1.1155813470094apolipoprotein L, 5155913477253Unknown156013487904Unknown156113489087serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1;protease inhibitor 2 (anti-elastase)156213489095sialoadhesin precursor; sialic acid-binding immunoglobulin-like lectin 1156313491972liver nuclear protein156413507059ubiquitin protein ligase156513509322suppression of tumorigenicity 5156613514831DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 10, ATP-dependent RNAhelicase156713516379aldehyde oxidase 1156813518228methylcrotonoyl-Coenzyme A carboxylase156913528660ribosomal protein L4, similar to157013528960ND 18k157113529047transforming growth factor, alpha157213529221PTD017 protein157313529257aldo-keto reductase family 1, member B1157413537192SCCA1b157513540475serum amyloid A2157613540477wingless-type MMTV integration site family, member 3 precursor157713540574Unknown157813540576Unknown157913540590C/EBP-induced protein158013540606suppressor of potassium transport defect 3 g158113543342Unknown158213543446Unknown158313543618ATPase B F0158413543706Unknown158513543933Unknown158613544007Unknown158713544072glycerol-3-phosphate dehydrogenase 1 (soluble), similarity to158813559241Unknown158913559363mitochondrial ribosomal protein L9159013559404mitochondrial ribosomal protein L43159113560110Unknown159213569848cell cycle progression 2 protein159313569913exonuclease NEF-sp159413569930toll-like receptor 10159513569948Unknown159613569962small GTP-binding protein159713591536Unknown159813606056DNA dependent protein kinase catalytic subunit159913620885mitochondrial ribosomal protein S6160013623251transcription factor EB, similar to160113623369Unknown160213623465peroxisomal long-chain acyl-coA thioesterase160313623483lysosomal-associated membrane protein 1160413623595DNA segment on chromosome 191177 expressed sequence160513623615Unknown160613623617Unknown160713623635Unknown160813623689Unknown160913623693Unknown161013626125ADAM-TS-9 precursor (A disintegrin and metalloproteinase withthrombospondin motifs 9) (ADAM-TS 9) (ADAM-TS9)161113627233aldo-keto reductase family 7, member A3161213627252oxoglutarate dehydrogenase161313627389elongation factor-2 kinase161413627804acyl-Coenzyme A dehydrogenase, short/branched chain precursor161513628614Na, K-ATPase subunit alpha 2161613628881Unknown161713629150cox 4161813630128faciogenital dysplasia protein161913630492Unknown162013630567Unknown162113630862aldehyde dehydrogenase 5 family, member A1162213630871Unknown162313630873protein kinase, cAMP-dependent, regulatory, type II, beta162413631242reelin162513631440PEROXIREDOXIN 2162613631521mitochondrial ribosomal protein S7162713631678UCR 5162813631907mitogen-activated protein kinase kinase kinase kinase 1162913632179myosin, heavy polypeptide 13, skeletal muscle163013632266thyroid hormone receptor interactor 2; PPARG binding protein163113632616carrier ANT2163213632896phosphoglucomutase 1163313633168plastin 3 precursor163413633370Notchhomolog 3163513635754CTCL tumor antigen se1-1163613635919Unknown (now 4507953)163713636042Unknown1638136360473-hydroxyisobutyryl-Coenzyme A hydrolase163913636157Unknown164013636168eukaryotic translation elongation factor 1 beta 2164113636504interferon-induced protein 75, 52 kD164213636598Unknown164313637083Unknown164413637529Unknown164513637537ETAA16 protein164613637608ND 75 K164713637631VDAC-2 voltage-dependent anion channel 2 (H. sapiens), similar to164813637711glycine cleavage system protein H (aminomethyl carrier) (H. sapiens),similar to164913637735Unknown165013637796Unknown165113637833cox 7a like, COX7RP (cytochrome c oxidase subunit VII-related protein),estrogen receptor binding CpG island165213637948glutathione S-transferase M5165313638591Unknown165413638758Unknown165513639105Unknown165613639114succinate dehydrogenase, Ip165713639187Unknown165813639470Unknown165913639628acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme Athiolase), mitochondrial166013639817malic enzyme 3, NADP(+)-dependent, mitochondrial166113640712phosphoinositide-3-kinase, class 2, alpha polypeptide166213640950interleukin 11 receptor, alpha166313641918sirtuin 3166413643253kinesin family member 3A166513643321Unknown166613643514Unknown166713643534ribosomal protein L12; 60S ribosomal protein L12 (H. sapiens), similar to166813643564exostoses 1166913643652flavohemoprotein b5 + b5R167013643704protein tyrosine phosphatase, receptor type167113644108proteasome 26S subunit, non-ATPase, 1167213644418Unknown167313644786butyrophilin, subfamily 1, member A1167413645381HLA-B associated transcript 2 (H. sapiens), similar to167513645492heat shock 70 kD protein-like 1167613645618dihydropyrimidinase related protein-3167713646385creatine kinase, sarcomeric mitochondrial167813646774quinoid dihydropteridine reductase167913647276L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain168013647558carrier ANT1168113647920gamma-glutamyltransferase 1168213647960tumor necrosis factor, alpha-induced protein 2168313648234Unknown168413648426cox assembly protein isoform 2168513648611serine/threonine kinase 2168613648964alanyl-tRNA synthetase168713649010odzhomolog 1168813649058Unknown168913649119SEX gene169013649217VDAC-1169113649475Unknown169213649658UCR ubiquinol-cytochrome c reductase binding protein169313650446heat shock 70 kD protein 2169413650574glutamate dehydrogenase 2 mitochondrial precursor169513650639melanoma antigen, family B, 1169613650785spectrin, beta, non-erythrocytic 5169713650793elongation factor SIII p15 subunit169813650874putative receptor protein169913650942Unknown170013650992Unknown170113651038leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIMdomains), member 4170213651229Rho GTPase activating protein 6 isoform 4170313651413Fc fragment of IgG binding protein (H. sapiens), similar to170413651526androgen-induced prostate proliferative shutoff associated protein170513651706golgin-like protein170613651985type 1 RNA helicase pNORF1 or nonsense-mediated mRNA decaytrans-acting factor170713652204Unknown170813652240ribosomal protein S7170913652246ARF protein171013652324ras-related small GTPasehypothetical protein X171113652801Rap1 guanine-nucleotide-exchange factor directly activated by cA171213653049acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor171313653910carboxypeptidase D precursor171413654274Unknown171513654278Unknown171613654294Unknown171713654678Unknown171813654685ATP-binding cassette, sub-family C, member 1, isoform 6171913655145UCR ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide-like 1172013655148EH-domain containing 2; EH domain containing 2, similar to172113655297Unknown172213676336Unknown172313676857heat shock 70 kD protein 2; Heat-shock 70 kD protein-2172413699811WHSC1L1 protein isoform long; Wolf-Hirschhorn syndrome candidate 1-like 1 protein172513751974Unknown172613774961autoimmune infertility-related protein172713775158Unknown172813775166Unknown172913775186ring finger protein 17 isoform long173013775208Unknown173113775210Unknown173213775216Unknown173313775232Unknown173413784938Unknown173513786129RAS-RELATED PROTEIN RAB-33B173613786847L-Lactate Dehydrogenase H Chain, Ternary Complex With Nadh AndOxamate173713787197DEAD/Hbox polypeptide 11173813787215sirtuin 5, isoform 2173913787217FAT tumor suppressor 2 precursor; multiple epidermal growth factor-likedomains 1; cadherin family member 8174013794267RAB7, member RAS oncogene family; Ras-associated protein RAB174113872241ligand of numb-protein X174213874437cerebral protein-11174313876386epiplakin 1174413899231mitochondrial ribosomal protein L9174513899275Unknown174613929460PTH-responsive osteosarcoma B1 protein174713929467chemokine binding protein 2174813937401Unknown174913937769RIKEN cDNA 1200013F24 gene, similar to175013937888heterogeneous nuclear ribonucleoprotein C175113938170Unknown175213938215taxol resistant associated protein175313938297heat shock cognate 71-kd protein, similar to175413938442neuronal protein, mitochondrial Complex I subunit175513938539cyclin D binding Myb-like transcription factor 1175613938571Unknown175713938593Unknown175813938619creatine kinase, muscle175913994164Charcot-Marie-Tooth duplicated region transcript 1176013994188AKAP-associated sperm protein176113994259mitochondrial ribosomal protein S5176213994280complement-c1q tumor necrosis factor-related protein 7 + F792176313994325putative b,b-carotene-9′, 10′-dioxygenase176414017783Unknown176514017783Unknown176614017807Unknown176714017833Unknown176814017865Unknown176914017899Unknown177014017903Unknown177114017903Unknown177214017923Unknown177314017941Unknown177414017943Unknown177514017949Unknown177614017957Unknown177714017971Unknown177814028389mitochondrial ribosomal protein L41177914028403mitochondrial ribosomal protein S28178014028405mitochondrial ribosomal protein S29178114028875UDP-glucuronic acid/UDP-N-acetylgalactosamine dual transporter;KIAA0260 protein; UDP-glucuronic acid/UDP-N-acetylgalactosamine dualtransporter178214028877mitochondrial ribosomal protein S25; mitochondrial 28S ribosomal proteinS25178314041699ESTRADIOL 17 BETA-DEHYDROGENASE 8178414041874MAPKK like protein kinase/PDZ-binding kinase178514041889Unknown178614041976Unknown178714041978CDA02 protein178814041989Unknown178914042018Unknown179014042066Unknown179114042110Unknown179214042216Unknown179314042323Unknown179414042336Unknown179514042441Unknown179614042814Unknown179714042822Unknown179814042850Unknown179914042923chromosome 9 open reading frame 5180014043187aldehyde dehydrogenase 4 A1180114043217plectin 1, intermediate filament bindi180214043281leucine-rich neuronal protein180314043412Unknown180414043451succinyl-CoA synthetase beta subunit GTP-specific180514043654phosphofructokinase, muscle, similar to180614043666Unknown180714043738Unknown180814124942ribophorin I, similar to180914124976kinesin family member C3181014133213Unknown181114133215Unknown181214133217Unknown181314133235Unknown181414141157heterogeneous nuclear ribonucleoprotein H3, isoform a181514149607chloride channel 7; CIC-7181614149625ND 20 k181714149649siah binding protein 1; FBP interacting repressor; pyrimidine tract bindingsplicing factor; Ro ribonucleoprotein-binding protein 1181814149677lectomedin-3181914149686Unknown182014149690Unknown182114149769GAJ protein182214149789Unknown182314149904tumor endothelial marker 8, isoform 1 precursor; anthrax toxin receptor182414149971Unknown182514150001Unknown182614150017Unknown182714150039Unknown182814150062Unknown182914150072Unknown183014150072Unknown183114150080Unknown183214150116Unknown183314150128phosphodiesterase 5A183414150134Unknown183514150155Unknown183614165260Unknown183714165270mitochondrial ribosomal protein L13183814192943MEGF10 protein183914194461A kinase anchor protein 9184014196457protocadherin gamma subfamily A, 12, isoform 2 precursor; cadherin 21;fibroblast cadherin FIB3184114196465protocadherin gamma subfamily A, 3, isoform 2 precursor184214198176ND 51 k184314198272Bcl-XL-binding protein v68, similar to184414198303Unknown184514211536neurexin 2; neurexin II184614211570conserved ERA-like GTPase184714211720desmuslin184814211857Unknown184914211903ubiquitin specific protease185014211907zinc finger protein 347; zinc finger 1111185114211923PKCI-1-related HIT protein185214211939methylmalonyl-CoA epimerase185314248761cAMP-specific cyclic nucleotide phosphodiesterase185414249144RAB11B, member RAS oncogene family185514249338Unknown185614249342internexin neuronal intermediate filament protein, alpha; neurofilament 5(66 kD); neurofilament-66, tax-binding protein185714249376Unknown185814249428Unknown185914249446Unknown186014249454Unknown186114249474Unknown186214249506Unknown186314249588lactamase, beta186414249596Unknown186514249620Unknown186614249967staufenhomolog 2186714250063peroxiredoxin 3186814250110Unknown186914250319Unknown187014250458stromal cell derived factor 5, similar to187114250628Unknown187214250744Unknown187314251209chloride intracellular channel 1187414269578metallothionein IV187514277739Erythrocyte Band-3 Protein, Crystal Structure Of The Cytoplasmic DomainOf Human187614280050Vps39/Vam6-like protein187714285174elongation factor G187814286186ZINC FINGER PROTEIN 185(P1-A) g187914286294Unknown188014289323LIP isoform of BLIP188114318622Unknown188214329511bA430M15.1 (novel protein (ortholog of rat four repeat ion channel))188314329531Unknown188414336727Unknown188514336768Unknown188614336775ND PDSW188714349362major histocompatibility complex, class I, F188814424013WNT-5B protein precursor188914424776Unknown189014485049T-cell receptor V delta 1189114488680Phosphoglucose IsomeraseNEUROLEUKINAUTOCRINE MOTILITYFACTORMATURATION Factor189214530763citrate lyase, similar to189314549163Matrilin-2 precursor189414571713tonicity-responsive enhancer binding protein189514575679hemicentin189614602477DNA-BINDING PROTEIN A189714602507Unknown189814602841cysteine string protein 1189914602856Unknown190014602907Unknown190114602977Unknown190214603084putative DNA binding protein190314603309heat shock 60 kD MITOCHONDRIAL190414603403stomatin-like 2190514670360zinc finger protein 278, long C isoform; POZ-AT hook-zinc finger protein190614714447sorting nexin 7190714714514DIHYDROLIPOAMIDE DEHYDROGENASE-BINDING PROTEIN OFPYRUVATE DEHYDROGENASE COMPLEX190814714528Unknown190914715007Unknown191014719392cofilin 2191114720172Unknown191214720558succinate dehydrogenase, flavoprotein subunit191314721241low density lipoprotein-related protein-associated protein 1191414721350testicular protein kinase 2191514721365hypothetical protein, estradiol-induced191614721507serine/threonine kinase 18191714721966Unknown191814722003cadherin 12, type 21919147221933-hydroxybutyrate dehydrogenase192014722283Unknown192114722554Unknown192214722589mitochondrial ribosomal protein L22192314722898mitochondrial ribosomal protein S27192414723145acid phosphatase 1 isoform b192514723407Unknown192614723451mitochondrial ribosomal protein L20192714723531p25192814724042ASB-3 protein192914724206Unknown193014724379Unknown193114724557phosphatidylinositol glycan, class K193214724575Unknown193314724751phosphorylase, glycogen; brain193414724805Unknown193514725181lymphocyte antigen 75193614725399TNF-induced protein193714725420syntaxin 12193814725545RNA-binding protein regulatory subunit193914725791Unknown194014725848acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain194114726372Unknown194214726632Unknown194314726693Unknown194414726725Unknown194514726866Unknown194614727174leucine-rich PPR-motif containing194714727486succinate dehydrogenase, subunit D194814727827Unknown194914728081excision repair cross-complementing rodent repair deficiency195014728229phosphoinositide-3-kinase, regulatory subunit 4, p150195114728316natural killer cell receptor 2B4195214728439Unknown195314728817Unknown195414728839Unknown195514728858sterol carrier protein 2195614728945DMRT-like family B with proline-rich C-terminal, 1195714729172elastin microfibril interface located protein195814729475BCL9195914729487mast cell carboxypeptidase A3 precursor196014729783dihydrolipoamide branched chain transacylase196114730158TATA element modulatory factor 1196214730499Unknown196314730569adenylate cyclase 3196414730600Unknown196514730775hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme Athiolase/enoyl-Coenzyme A hydratase alpha196614730782kinesin heavy chain member 2196714732014Unknown196814732481calcium channel, voltage-dependent, alpha 1E subunit196914732525selective LIM binding factor, rat homolog197014732721adenomatosis polyposis coli197114732789mitofilin197214732886thyroid hormone receptor-associated protein, 150 kDa subunit197314733183adaptor-related protein complex 2, mu 1 subunit197414733451enkephalinase197514733480Unknown197614733532myofibrillogenesis regulator MR-1197714733712chondroitin sulfate proteoglycan 2197814733904serine/threonine kinase 16197914734022Unknown198014734151lymphoid enhancer binding factor-1198114734205Unknown198214734243Unknown198314734441Unknown198414734746DEAD/Hbox polypeptide 1198514734864SWI/SNF related, matrix associated, actin dependent regulator ofchromatin, subfamily a-like 1198614735060mitochondrial isoleucine tRNA synthetase198714735128Ste-20 related kinase198814735161BCL6198914735336Unknown199014735426nuclear factor, interleukin 3 regulated199114735687Unknown199214735741Unknown199314735899cytochrome b5 reductase 1199414736223UCR 1199514736227Rho-associated, coiled-coil containing protein kinase 2199614736267protein disulfide isomerase-related protein199714736397Unknown199814736560Unknown199914736612Unknown200014736678lactotransferrin200114736760voltage-dependent anion channel 2200214736866DnaJhomolog, subfamily B, member 12200314737445sema domain, immunoglobulin domain (lg), short basic domain,200414737746myeloid differentiation primary response gene200514737907Unknown200614738004Unknown200714738099Apobec-1 complementation factor; APOBEC-1 stimulating protein200814738103annexin IV200914738306putative, similar to201014738689Unknown201114738950Unknown201214739002Unknown201314739106Unknown201414739392Unknown201514739472potassium voltage-gated channel, shaker-related subfamily201614739880Unknown201714740316HEAT SHOCK 27 KDA PROTEIN (HSP 27) (STRESS-RESPONSIVEPROTEIN 27) (SRP27) (ESTROGEN-REGULATED 24 KDA PROTEIN)(28 KDA HEAT SHOCK PROTEIN), similar to201814740371A kinase anchor protein 2201914740403thioredoxin202014740476TAF2 RNA polymerase II, TATA box binding protein (TBP)-associatedfactor, 150 kD202114740547FUMARATE HYDRATASE, MITOCHONDRIAL PRECURSOR(FUMARASE)202214740792v-ral simian leukemia viral oncogene homolog A (ras related)202314740886Unknown202414741177Unknown202514741234Unknown202614741376Fas-activated serine/threonine kinase, isoform 2202714741510Unknown202814741555Unknown202914741636Unknown203014741782uncharacterized hematopoietic stem/progenitor cells protein MDSO203114742266RNA helicase203214742273Unknown203314742317Unknown203414742600vimentin203514742688diphthamide biosynthesis-like protein 2203614742977inter-alphainhibitor, H2 polypeptide203714743031Unknown203814743873TAR (HIV) RNA binding protein 1203914744078gamma filamin204014744132heat shock 70 kD protein 5 (glucose-regulated protein, 78 kD)204114744234nuclear receptor subfamily 6, group A, member 1, isoform 1204214744290Hermansky-Pudlak syndrome protein204314744642Unknown204414744702rat myomegalin, similar to204514745217lipocalin 2 (oncogene 24p3)204614745424spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)204714745489wingless-type MMTV integration site family, member 3A204814745808guanine nucleotide binding proteinalpha 12204914745853Z-band alternatively spliced PDZ-motif205014745861Z-band alternatively spliced PDZ-motif205114745865Unknown205214746475Unknown205314746487ACYL-COA DEHYDROGENASE, VERY-LONG-CHAIN SPECIFIC + F36,similar to205414746491Unknown205514746535RAB7, member RAS oncogene family205614746585yeast adenylate cyclase, similar to205714747216carrier aralar205814747249CGI-135 protein205914747260serologically defined colon cancer antigen 1206014747375lysophospholipase I206114747970Unknown206214748292Unknown206314748400Unknown206414748439Unknown206514748831Unknown206614748858transformation/transcription domain-associated protein206714749079vacuolar protein sorting protein 18206814749154Unknown206914749213serine-threonine kinase/MAD3-like protein kinase207014749294GCN2 elF2alpha kinase207114749361Unknown207214749419Unknown207314749523Unknown207414749588Unknown207514749765A kinase anchor protein 6207614749776Unknown207714750136Unknown207814750148Unknown207914750186LAMIN A/C (70 KDA LAMIN)208014750222Unknown208114750259Rho/Rac guanine nucleotide exchange factor 2208214750405pyruvate kinase, muscle (H. sapiens), similar to208314751203Unknown208414751493N-acylsphingosine amidohydrolase208514751551Unknown208614751705Unknown208714751808purine nucleoside phosphorylase208814751866IGF-II mRNA-binding protein 3208914752024carrier aralar2209014752229dihydrolipoamide dehydrogenase209114752236Unknown209214752239laminin, beta 1 precursor209314752249spectrin, beta, erythrocytic (includes spherocytosis, clinical type I)209414752728guanine nucleotide exchange factor Lbc or A-kinase anchoring protein209514753117Unknown209614753239kinectin 1209714753384A kinase (PRKA) anchor protein (gravin) 12209814753693adaptor-related protein complex 4, sigma 1 subunit, similar to209914753915Ras protein-specific guanine nucleotide-releasing factor 1210014754222farnesol receptor HRR-1210114754627Unknown210214754848Unknown210314754867Unknown210414755192Unknown210514755316zinc finger protein 91210614755336tumor rejection antigen 1210714755347Unknown210814755357mitochondrial ribosomal protein L18210914755436superoxide dismutase 2, mitochondrial211014755456zinc finger protein 256211114755952lysophospholipase I, similar to211214756295Na, K-ATPase subunit alpha 3211314756299pot. ORF (1013 AA), similar to211414756626DNA (cytosine-5)-methyltransferase211514756630mitochondrial ribosomal protein L4211614756895dUTP pyrophosphatase211714756939Unknown211814756944Unknown211914757147Unknown212014757210FSH primary responsehomolog 1212114757677phosphoglycerate kinase 1212214757711Unknown212314758001ND 24K NADH dehydrogenase (ubiquinone) flavoprotein 2 (24 kD) (H.sapiens), similar to212414758520ATPase, Cu++ transporting, beta polypeptide (Wilson disease)212514759302golgi autoantigen, golgin subfamily a, 3212614759459hook2 protein212714759609Unknown212814759903transcription factor212914759981Unknown213014760649inositol 1,4,5-triphosphate receptor, type 2213114761208glyceraldehyde 3-phosphate dehydrogenase like213214761398tubulin beta 5, similar to213314761496programmed cell death 8 (apoptosis-inducing factor)213414761689calcium channel, voltage-dependent, beta 3 subunit213514762250protein tyrosine phosphatase, receptor type, B213614762650Unknown213714762696granzyme M precursor213814763105Unknown213914763304src homology 2 domain-containing transforming protein D, similar to214014763427death-associated protein kinase 3, ZIP-kinase214114763491NY-REN-58 antigen214214763709Unknown214314763948FERM, RhoGEF, and pleckstrin domain protein 1; chondrocyte-derivedezrin-like protein, similar to214414764159acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-CoenzymeA thiolase)214514764202hydroxyacyl-Coenzyme A dehydrogenase, type II214614764412D-amino-acid oxidase214714764458male-specific lethal-3 (Drosophila)-like 1214814764705Unknown214914764874Unknown215014764936G protein-coupled receptor 19215114765579Unknown215214765581peroxiredoxin 5215314765684kinesin family member 4215414766197Unknown215514766265Unknown215614766346glutathione S-transferase P1-1215714766373regulatory factor X, 4215814766393transmembrane protein (63 kD), endoplasmic reticulum/Golgi215914766635prohibitin, B-cell associated protein216014766937DRIM protein or Key-1A6 protein216114767036Unknown216214767224protein kinase C and casein kinase substrate216314767305protein C, cardiac216414767738CALCIUM ATPASE 2(SERCA2)216514767795Unknown216614768227purinergic receptor P2X, ligand-gated ion channel, 7216714768743thioredoxin peroxidase216814769051ND B14.5a216914769064Unknown217014769085Unknown217114769089Unknown217214769268GalNAc alpha-2, 6-sialyltransferase I, long form217314769776peripheral benzodiazepine receptor-associated protein 1217414770042Unknown217514770070Unknown217614770170Unknown217714770383Unknown217814770569Unknown217914770608small fragment nuclease218014770670Unknown218114770915Unknown218214770940angiotensin I converting enzyme218314770968Unknown218414771355beta-2-glycoprotein I precursor218514771369brain-immunoglobulin-like molecule with tyrosine-based activation motifs218614771396isocitrate dehydrogenase 3 beta (NAD+)218714771416murine retrovirus integration site 1 homolog218814771689myosin, heavy polypeptide 1, skeletal muscle, adult218914772046Unknown219014772333phosphorylase, glycogen; brain (H. sapiens), similar to219114772527Unknown219214772555Unknown219314772672calpain 5219414772954copine I219514773504tyrosine kinase, non-receptor, 1219614773592AHNAK nucleoprotein (desmoyokin)219714773948Unknown219814774045Unknown219914774139ATPase g220014774236Unknown220114774282apolipoprotein A-I precursor220214774359ionotropic ATP receptor P2X5b220314774503phospholipase D2220414774525carrier oxoglutarate220514774778Unknown220614774780karyopherin (importin) beta 1220714774844succinate dehydrogenase, subunit C220814775218Unknown220914775320Unknown221014775363baculoviral IAP repeat-containing protein 5221114775444carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5, similar to221214775476endocytic receptor (macrophage mannose receptor family)221314775546malonyl-CoA decarboxylase221414775827ubiquinol-cytochrome c reductase core protein II221514775827UCR 2221614776296Unknown221714776472nuclear receptor co-repressor 1221814776681Unknown221914776736Unknown222014776778ATP-binding cassette, sub-family A member 3222114776800cat eye syndrome chromosome region, candidate 5, isoform 1222214776960Unknown222314776980carrier citrate transporter222414777215protein disulfide isomerase, pancreatic; protein disulfide isomerase,similar to222514777313ND 13 k-B222614777483general transcription factor IIIC, polypeptide 1 (alpha subunit, 220 kD)222714777522Unknown222814777630AT-binding transcription factor 1222914777716Unknown223014777813Unknown223114777901Unknown223214778035Unknown223314778104adaptor-related protein complex 1, beta 1 subunit223414778235Unknown223514778381eIF4E-transporter223614778431ret finger protein-like 2223714778654THIOSULFATE SULFURTRANSFERASE (RHODANESE)223814779326Unknown223914779686Unknown224014779867N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminida224114779881periplakin224214779964Unknown224314780055protease, serine, 7224414780117Unknown224514780193synaptojanin 1224614780272intersectin 1 (SH3 domain protein)224714780668ES1 protein /KNP-I protein ?? (ThiJ/Pfpl family motif)224814780705phosphofructokinase, liver224914780857Unknown225014781094huntingtin225114781125quinoid dihydropteridine reductase (H. sapiens), similar to225214781245fatty-acid-Coenzyme A ligase, long-chain 6225314781533Unknown225414781826receptor (TNFRSF)-interacting serine-threonine kinase 1225514781890Unknown225614781979Unknown225714781989putative transcription factor/GTF2I repeat domain-containing 1, isoform 2225814782063malate dehydrogenase 2, NAD (mitochondrial)225914782332HLA-B associated transcript-3, similar to226014782751Unknown226114782921protein kinase C and casein kinase substrate in neurons 1226214782973tubby like protein 1226314783011p38 mitogen-activated protein kinase226414783112Unknown226514783333supervillin, isoform 1226614783455Unknown226714783504Unknown226814783675small GTP binding protein RAB6 isoform226914783738inositol polyphosphate phosphatase-like 1227014784011Unknown227114784064mitogen-activated protein kinase kinase kinase 11227214784122atrophin-1227314784162Ubiquitin isopeptidase T227414784612Unknown227514784913EH-domain containing 4227614785008Unknown227714785181microfibrillar-associated protein 1227814785356Unknown227914785405polo (Drosophia)-like kinase228014785865Unknown228114785919copper containing amine oxidase 3 precursor; amine oxidase (copper-containing); copper amine oxidase precursor; vascular adhesion protein 1;vascular adhesion protein 1, similar to228214786231Unknown228314786366PAR-6 beta228414786394cytochrome P450, subfamily XXIV precursor228514786884Unknown228614787181CUB and sushi multiple domains protein 1 short form228714790190SMART/HDAC1 associated repressor protein228815012003Unknown228915012048HERV-H LTR-associating 3, similar to229015020655ATP/GTP-binding protein229115026974obscurin229215029619fracture callus 1 homolog229315029922Unknown229415030240ATPase alpha, H+ transporting, mitochondrial F1 complex, alpha subunit,isoform 1, cardiac muscle, similar to229515041811Hermansky-Pudlak syndrome type-3 protein229615076827Pcph proto-oncogene protein229715079348angiotensinogen proteinase inhibitor,229815079392replication control protein 1229915079408Unknown230015079735Unknown230115080291dipeptidyl peptidase 7+F206, similar to230215080429Unknown230315080454Unknown230415080499serineproteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 1, similar to230515126735heat shock 27 kD protein 1, similar to230615147248putative breast epithelial stromal interaction protein230715147337progestin induced protein; ubiquitin-protein ligase [Homo sa230815149476arginyl-tRNA synthetase230915150811mitochondrial ribosomal protein S36231015208648central cannabinoid receptor, isoform b; CB1 receptor; brain cannabinoidreceptor 1231115213479putative DNA polymerase delta p38 subunit231215213542NSD1231315214423Unknown231415214486Unknown231515214706Unknown231615215308dystroglycan 1, similar to231715227456ch-TOG protein from Homo sapiens [Arabidopsis tha231815277229Homologue to Drosophila photoreceptor protein calphotin231915277415scavenger receptor cysteine-rich type 1 protein M160 precursor232015277514Unknown232115278188Unknown232215281150unkempt (Drosophila)-like232315281837PX domain-containing protein kinase232415294558RAS-RELATED PROTEIN RAB-5A232515294560RAB5A, member RAS oncogene family232615294667bassoon (presynaptic cytomatrix protein)232715294817GalNAc-4-sulfotransferase 2 (H. sapiens), similar to232815295270MADhomolog 5232915295351VDAC-1233015295412Unknown233115295574laminin receptor1233215295842Unknown233315296104optic atrophy 1233415296351splicing factor 3b, subunit 1, 155 kD233515296762v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog233615296824lipin 1233715297926transforming growth factor, alpha233815298022mitochondrial ribosomal protein L53233915299136Unknown234015299287Unknown234115299581Unknown234215299784glutamate receptor, metabotropic 1234315299942Unknown234415300149modulator of transcription factor GATA-4 in cardiomyocytes234515301488SERINE/THREONINE PROTEIN KINASE 24(MST-3)234615302083CD2-associated protein234715302719Unknown234815302936citrate synthase precursor234915303880Glutamate receptor interacting protein235015304843Unknown235115304935destrin (actin depolymerizing factor)235215305404Unknown235315305472troponin I, cardiac235415305838ReIA-associated inhibitor235515306072transcriptional repressor NAC1235615306753Unknown235715307117rho guanine nucleotide exchange factor 12235815307634ND 23 k235915314651oxygen regulated protein236015318843aconitase 2, mitochondrial236115318933cytochrome b5 reductase236215321298Unknown236315321380v-erb-a avian erythroblastic leukemia viral oncogene homolog-like 4236415321446Unknown236515341707Unknown236615375094RSK-like protein236715451842ADAM-TS disintegrin and metalloproteinase domain 19, isoform 1preproprotein; meltrin beta; metalloprotease-disintegrin meltrin beta236815451854midline 1, isoform beta; midline-1; zinc finger X and Y236915451916bone morphogenetic protein receptor, type II, isoform 1 precursor; type IIactivin receptor-like kinase; serine/threonine kinase237015451923serologically defined colon cancer antigen 33237115529996son of sevenless homolog 1 (Drosophila); son of sevenless (Drosophila)homolog 1237215530243villin 2 (ezrin), similar to237315530305Unknown237415553127hexokinase 2; hexokinase-2, muscle237515553137H2A-Bbd237615559225Unknown237715559303Unknown237815559516Unknown237915559753Unknown238015620821Unknown238115620841Unknown238215620853Unknown238315620867Unknown238415620879Unknown238515620927Unknown238615620933Unknown238715680004H2B histone family, member Q, similar to238815680171semaF cytoplasmic domain associated protein 3238915718530POM121 membrane glycoprotein (rat homolog)-like 2239015778991Unknown239115779080Unknown239215779126guanine nucleotide binding protein (G protein), a239315779156Unknown239415795410Unknown239515808373erythroid membrane-associated protein239615808607ATPase f F0239715826629Peroxiredoxin 5239815928608solute carrier family 25 (mitochondrial carrier; adenine nucleotidetranslocator), member 5, similar to239915928907Unknown240015929030Unknown240115929352mitochondrial ribosomal protein L1240215929856Unknown240315929892Unknown240415988268Myb-Domain Of Human Rap1240515988350Lysozyme240615990494Unknown240715991827hexokinase 1, isoform HKI-R; brain form240815991829hexokinase 1, isoform HKI-ta/tb; brain form hexokinase240915991859Unknown241016033591SH2 domain-containing phosphatase anchor protein 2b241116041807Unknown241216156815Sec23-interacting protein p125241316156952Unknown241416157047succinate dehydrogenase complex, subunit A, flavoprotein precursor241516157111progesterone membrane binding protein241616157253uridine 5 monophosphate hydrolase 1; pyrimidine 5-nucleotidase, similarto241716157453Unknown241816157682IDN3 protein241916158005RNA-binding protein regulatory subunit242016158038putative, similar to242116158324heat shock 70 kD protein (Mortalin-2)242216158747CLIP-associating protein 2242316159170Unknown242416159302Unknown242516159416Unknown242616159569Unknown242716159594carnitine palmitoyltransferase II242816159701ribosomal protein S7 (H. sapiens), similar to242916159788S100 calcium-binding protein A6243016159874Unknown243116160276spectrin, beta, non-erythrocytic 1 (H. sapiens), similar to243216160441putative, similar to243316160793glycosyltransferase AD-017243416160823phosphatidylinositol-4-phosphate 5-kinase, type I, beta243516160929retinoblastoma-binding protein 5243616161569ryanodine receptor 2243716161583endoplasmic reticulum oxidoreductin 1-Lbeta243816161627Rho guanine nucleotide exchange factor 10243916161681Unknown244016161727stromal cell derived factor receptor 1 isoform a244116162032PEPTIDYL-PROLYL CIS-TRANS ISOMERASE B PRECURSOR(PPIASE) (ROTAMASE) (CYCLOPHILIN B)244216163057Unknown244316163065RIKEN cDNA 2410008H17 gene, similar to244416163124TTF-I interacting peptide 20244516163817Bcl 1244616164710Unknown244716164895rabaptin-5244816164980Unknown244916165190Unknown245016165554Unknown245116165872accessory proteins BAP31/BAP29 (H. sapiens), similar to245216166325Unknown245316166513pericentrin B245416168619Unknown245516171486Unknown245616171987monoamine oxidase A245716172349triadin245816174655Unknown245916175846atrophin-1 interacting protein 1; activin receptor interacting protein246016176937excision repair protein 1246116177368putative, similar to246216177559MLL2 protein246316178062Unknown246416178117Unknown246516178214GTP-rho binding protein 1, similar to246616181084G protein-coupled receptor 51246716192638isocitrate dehydrogenase 2 (NADP+), mitochondrial246816196598cox 6a246916198361Unknown247016198481Unknown247116306537cadherin 20, type 2 preproprotein247216306954Unknown247316306978annexin A2247416307164CGI-90 protein247516307227Unknown247616307270Unknown247716307468Unknown247816307475neuroepithelial cell transforming gene 1247916359102Unknown248016359195Unknown248116416451tRNA-nucleotidyltransferase248216418373Unknown248316418423guanylate binding protein 4248416507813tumor necrosis factor receptor superfamily, member 21, similar to248516549125Unknown248616549199Unknown248716549271Unknown248816549294Unknown248916549620Unknown249016549880Unknown249116549918Unknown249216550394Unknown249316550518Unknown249416550576Unknown249516550810Unknown249616550845Unknown249716551173Unknown249816551429Unknown249916551580Unknown250016551610Unknown250116551739myosin light chain kinase250216551769Unknown250316551917Unknown250416551953Unknown250516551957Unknown250616552104Unknown250716552271Unknown250816552547Unknown250916552885Unknown251016552927Unknown251116552957Unknown251216552988Unknown251316553031Unknown251416553078Unknown251516553235Unknown251616553285Unknown251716553362Unknown251816554014Unknown251916554275Unknown252016554604mitochondrial ribosomal protein S23252116554607mitochondrial ribosomal protein S10; NB4 apoptosis/differentiation relatedprotein; mitochondrial 28S ribosomal protein S10252216741033protease 26S subunit, ATPase 1252316753264Unknown252416876860Unknown252516877071ATPase gamma F1252616877127synaptophysin-like protein, similar to252716877285duodenal cytochrome b, similar to252816877328Unknown252916877328Unknown253016877459Unknown253116877964isovaleryl Coenzyme A dehydrogenase253216878101Unknown253316924265Unknown253416924269Unknown253516950603mitochondrial ribosomal protein S35; mitochondrial 28S ribosomal proteinS28253616950609mitochondrial ribosomal protein S27; mitochondrial 28S ribosomal proteinS27253716974753sodium-potassium-chloride cotransporter253817016315olfactory receptor-like protein JCG4253917028367gelsolin (amyloidosis, Finnish type), similar to254017028379Unknown254117375734Cyclin G-associated kinase254217378599Gamma-interferon-inducible protein Ifi-16 (Interferon-inducible myeloiddifferentiation transcriptional activator) (IFI 16)254317380287Mitochondrial 39S ribosomal protein L56 (MRP-L56) (Serine betalactamase-like protein LACTB)254417380426Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA (Processing alpha-1,2-mannosidase IA) (Alpha-1,2-mannosidase IA) (Mannosidase alphaclass 1A member 1) (Man(9)-alpha-mannosidase) (Man9-mannosidase)254517389971Unknown254617402865thiosulfate sulfurtransferase (rhodanese)254717432231MSTP022254817434094putative, similar to254917434314Unknown255017434411Unknown255117434458Unknown255217434554Unknown255317434671Unknown255417435264INNER EAR-SPECIFIC COLLAGEN PRECURSOR (SACCULARCOLLAGEN), similar to255517435299Unknown255617435748phosphorylase, glycogen; brain255717436258ND 13 K-B NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5;hypothetical protein FLJ12147; Complex I-13 KD-B; ubiquinone reductase;type I dehydrogenase, similar to255817436498Unknown255917436513VDAC-1 VOLTAGE-DEPENDENT ANION-SELECTIVE CHANNELPROTEIN 1 (VDAC-1) (RVDAC1) (OUTER MITOCHONDRIALMEMBRANE PROTEIN PORIN 1), similar to256017436561Unknown256117436979Unknown256217437312Unknown256317438284Unknown256417439551REGULATOR OF G-PROTEIN SIGNALING 12 (RGS12), similar to256517440287anaplastic lymphoma kinase Ki-1, similar to256617442134one twenty two protein; hypothetical protein FLJ12479, similar to256717442500Molybdenum cofactor synthesis protein cinnamon, similar to256817442568Unknown256917443010hematological and neurological expressed sequence 1, similar to257017443439Unknown257117443833glyceraldehyde-3-phosphate dehydrogenase, similar to257217444067RIKEN cDNA 0610011N22, similar to257317444600Unknown257417444969solute carrier family 4, anion exchanger, member 3257517445877xylulokinase homolog (H. influenzae)257617446038Unknown257717446807plastin 1257817447126Unknown257917447383Unknown258017447877Unknown258117450039Unknown258217450491factor V, similar to258317451676putative, similar to258417451748Unknown258517451801Unknown258617452377Unknown258717454350putative protein, similar to258817454582phosphoglycerate mutase 1 (brain); Phosphoglycerate mutase A,nonmuscle form, similar to258917455099putative, similar to259017455439heat shock 60 kD protein 1 (chaperonin) (H. sapiens), similar to259117455445Mitochondrial Complex I protein, now 21754001259217455927Unknown259317456092Unknown259417456384non-specific cross reacting antigen, similar to259517457389Unknown259617458483Unknown259717458911Unknown259817459115Melanoma-associated antigen 11 (MAGE-11 antigen), similar to259917459319putative, similar to260017459408small Rho-like GTPase RhoA, similar to260117459479Unknown260217459746VOLTAGE-DEPENDENT ANION-SELECTIVE CHANNEL PROTEIN 2(OUTER MITOCHONDRIAL MEMBRANE PROTEIN PORIN 2), similar to260317460020Unknown260417460330Unknown260517460767Unknown260617460836testis expressed sequence 13A, similar to260717461025Unknown260817461670RIKEN cDNA 9430083G14, similar to260917462761Unknown261017463437Unknown261117464527match: multiple proteins; match: Q08151 P28185 Q01111 Q43554;match: Q08150 Q40195 P20340 Q39222; match: Q40368 P36412P40393 Q40723; match: CE01798 Q38923 Q40191 Q41022; match:Q39433 Q40177 Q40218 Q08146; match: P10949 P11023 Q, similar to261217464573Unknown261317464724eukaryotic translation elongation factor 1 alpha 1, similar to261417464807phosphoglycerate mutase 2 (muscle)261517464864Unknown261617465135v-raf murine sarcoma viral oncogene homolog B1261717465213Unknown261817465562Unknown261917466365Unknown262017466818Unknown262117468096prohibitin, similar to262217468798Unknown262317469624Unknown262417470256Unknown262517470269chromosome 15 open reading frame 2, similar to262617470290Unknown262717471316Unknown262817471893Unknown262917472555Unknown263017472883ND 51K NADH dehydrogenase (ubiquinone) flavoprotein 1 (51 kD)263117474293midline 1; Finger on X and Y (in rat only on X), similar to263217474785VDAC-1 voltage-dependent anion channel 1, similar to263317475184Y39B6A.pp.p, similar to263417476245Unknown263517476469Unknown263617476471Unknown263717478738Unknown263817481443procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), beta polypeptide (protein disulfide isomerase; thyroidhormone binding protein p55)263917481778Unknown264017482059Unknown264117482696Kruppel-type zinc finger (C2H2), similar to264217482910Unknown264317482953putative methyl-binding domain protein MBD105, similar to264417483121rhophilin-like protein (H. sapiens), similar to264517483187Unknown264617483399RAB11B, member RAS oncogene family264717483482Unknown264817484820acetyl-Coenzyme A synthetase 2 (AMP forming)-like264917484835Unknown265017485036Unknown265117485099Unknown265217485128Unknown265317485337Unknown265417485700Unknown265517485787Mitochondrial Acyl-CoA Thioesterase265617486071DKFZP434O047 protein, similar to265717486087Unknown265817486456Unknown265917486463Unknown266017486622Unknown266117486915Unknown266217487175dentin phosphoryn, similar to266317487390Unknown266417487672Unknown266517487733F40G9.9.p, similar to266617487809glyceraldehyde-3-phosphate dehydrogenase, similar to266717487981F4N2.10, similar to266817488153Unknown266917489631Unknown267017491107Unknown267117511874Unknown267217511976Unknown267317512080WAS protein family, member 1267417512147Unknown267517736731mixed lineage kinase 4beta267617834080haymaker protein267717865554mitochondrial ribosomal protein L9, 60S mitochondrial precursor (L9 mt)267817939563Unknown267917943068Tcf-4 BETA-Catenin Complex268017943407Auh Protein, An Rna-Binding Homologue Of Enoyl-Coa Hydratase268117981863ND 5268217985539ND 4268318044194Unknown268418087815Unknown268518088572RIKEN cDNA 4930553C05 gene, similar to268618147097CG1800 gene product [Drosophila melanogaster] homolog268718157651bullous pemphigoid antigen 1 eA268818158416chromosome 20 open reading frame 188 protein; likely ortholog of mousetransient receptor protein 4, associated protein268918201886chromosome 20 open reading frame 175269018201913winged-helix nude269118204214Unknown269218204272Unknown269318252315propionyl-CoA carboxylase alpha subunit269418252778ankyrin repeat-containing protein ASB-2269518490293ephrin B3, similar to269618490363calsequestrin 2 (cardiac muscle)269718490389Unknown269818490639Unknown269918543654Unknown270018543672Unknown270118544062Unknown270218544103transcription factor Dp-1, similar to270318544502Unknown270418545149SWI/SNF related, matrix associated, actin dependent regulator ofchromatin, subfamily f, member 1 (H. sapiens), similar to270518545197Unknown270618545286Unknown270718545525Unknown270818545711trithorax-related, similar to270918545867forkhead box D2271018546369Unknown271118546495N-acetylglucosaminyltransferase VI, similar to271218547145Unknown271318547604Unknown271418547655Unknown271518547774PAPIN, similar to271618547995Unknown271718548319Unknown271818548686Unknown271918548841Unknown272018549011Unknown272118549603Unknown272218549721spectrin, alpha, erythrocytic 1 (elliptocytosis 2)272318549759Unknown272418550245Unknown272518550248dysferlin272618550356Unknown272718550688LWamide neuropeptide precursor protein, similar to272818551342laminin receptor 1; Laminin receptor-1 (67 kD); 67 kD, ribosomal proteinSA, similar to272918551404Unknown273018551428Unknown273118551530Unknown273218551750Unknown273318552428down-regulated by Ctnnb1, a, similar to273418552574heat shock 70 kD protein 9B (mortalin-2) (H. sapiens), similar to273518552843Unknown273618553054Unknown273718553524Unknown273818553646Unknown273918553709RIKEN cDNA 1810055D05 gene, similar to274018553922succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (H.sapiens) similar to274118554092Unknown274218554792Unknown274318554892protein phosphatase 4 regulatory subunit 2 (H. sapiens), similar to274418555498Unknown274518555697SALL1 (sal (Drosophila)-like, similar to274618555923Unknown274718556527protein tyrosine phosphatase, receptor type, G274818557013Unknown274918557341Unknown275018557515ring finger protein 23; RING-B box-coiled coil-B30.2, similar to275118557535Unknown275218557606Unknown275318557689Unknown275418558040Unknown275518558112C-terminal binding protein 1 (H. sapiens), similar to275618558130cyclin G associated kinase (H. sapiens), similar to275718558177Unknown275818558348Unknown275918558362Unknown276018558762Unknown276118559050Unknown276218559054Unknown276318559169GrpE-like protein cochaperone276418559889Unknown276518559896Unknown276618559969Unknown276718559997Unknown276818560088Unknown276918560396Unknown277018560536Unknown277118560871Unknown277218560910SGC32445 protein277318561153Unknown277418561225Unknown277518561342Unknown277618561850Unknown277718562164Unknown277818562264Unknown277918562403gag, similar to278018562447Unknown278118562613Unknown278218562676Unknown278318562743Unknown278418562778Unknown278518562814Unknown278618562826Unknown278718563024Unknown278818563079Unknown278918563446Unknown279018564249Unknown279118565200Unknown279218565553Unknown279318565735Unknown279418565792Unknown279518565965Unknown279618566008Unknown279718566051Unknown279818566469CDC14 cell division cycle 14 homolog B (S. cerevisiae) (H. sapiens),similar to279918566582Unknown280018567546Unknown280118568015Unknown280218568092Unknown280318568100Unknown280418568732Unknown280518568834Unknown280618568892T-COMPLEX PROTEIN 1, GAMMA SUBUNIT (TCP-1-GAMMA) (CCT-GAMMA), similar to280718568988Unknown280818569016Unknown280918569389Unknown281018569391Unknown281118569544Unknown281218569728Unknown281318569926Unknown281418570016Unknown281518570037Unknown281618571373Unknown281718571864Unknown281818572080tubulin, beta polypeptide 4, member Q (H. sapiens), similar to281918572219Unknown282018572532Unknown282118572576DKFZP434J193 protein (H. sapiens), similar to282218572752Unknown282318573432Unknown282418573604Unknown282518573884Sec24-related protein C282618574091(H. sapiens), similar to282718574564Unknown282818574897cathepsin L, similar to282918575014Unknown283018575020Unknown283118575034Unknown283218575353Unknown283318575792Unknown283418575881solute carrier family 9 (sodium/hydrogen exchanger), isoform 3, similar to283518575937Unknown283618576372Unknown283718576435glycoprotein beta-Gal 3′-sulfotransferase (H. sapiens), similar to283818576618Unknown283918576708Unknown284018576758Unknown284118576861Unknown284218577160Unknown284318577199suppression of tumorigenicity 5284418577427Unknown284518577553Unknown284618577877glutamate receptor, metabotropic 5 (H. sapiens), similar to284718578024Unknown284818578981voltage gated potassium channel Kv3.2b, similar to284918579037glyceraldehyde-3-phosphate dehydrogenase, similar to285018579791Unknown285118580015Unknown285218580073Unknown285318580116solute carrier family 4, sodium bicarbonate cotransporter, member 8 (H.sapiens), similar to285418580149Unknown285518580193Unknown285618580223Unknown285718580396Unknown285818580585Unknown285918580633phosphoinositide-3-kinase, class 2, gamma polypeptide286018581005Unknown286118581215Unknown286218581598Unknown286318581873Unknown286418582200Unknown286518582274Unknown286618582343Unknown286718582592Unknown286818582682CG9109 gene product, similar to286918582865Unknown287018583213Unknown287118583325Unknown287218583345Unknown287318583383Unknown287418583657Unknown287518583725multidomain presynaptic cytomatrix protein Piccolo, similar to287618583727Unknown287718584065Unknown287818584949Unknown287918585335Unknown288018585686Unknown288118586054Unknown288218586298Unknown288318586333splicing factor 3b, subunit 3, 130 kD288418586459putative, similar to288518586610Unknown288618587004Unknown288718587044Unknown288818587067Unknown288918587111Unknown289018587387Unknown289118587810arachidonate 12-lipoxygenase, 12R type (H. sapiens), similar to289218588235Unknown289318588450Unknown289418588517Unknown289518589035Unknown289618589065WW domain binding protein-2, similar to289718589260Unknown289818589408Unknown289918589876Unknown290018590023Unknown290118590390RNI-like protein, similar to290218590417Unknown290318590816Unknown290418591174Unknown290518591441ND B14.5a NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7(14.5 kD, B14.5a)290618591813Unknown290718592023Unknown290818592069Unknown290918592852Unknown291018593545Unknown291118593908Unknown291218593939secretory protein 45 kDa, similar to291318594017Unknown291418594189Unknown291518594359Unknown291618594592Unknown291718594594Unknown291818594767Unknown291918594954Unknown292018594992Unknown292118595043Unknown292218595057Unknown292318595318Unknown292418595340Unknown292518595665Unknown292618596319glycerol kinase (H. sapiens), similar to292718596413Unknown292818596484Unknown292918596861RAS-RELATED PROTEIN RAB-15, similar to293018597225Unknown293118597549ZINC FINGER PROTEIN 268 (ZINC FINGER PROTEIN HZF3), similar to293218597551Unknown293318597742Unknown293418598132Unknown293518598291kinesin family member C3293618598462Unknown293718598482Unknown293818598674Unknown293918598989Unknown294018599137zinc finger protein 2 (A1-5)294118599227Unknown294218599297EphB1294318599533polyhomeotic 2 protein, similar to294418599587Unknown294518600174Unknown294618600186Unknown294718600274Unknown294818600320Unknown294918600459axonal transport of synaptic vesicles295018600477Unknown295118600510Unknown295218600673replication initiation region protein (60 kD) (H. sapiens), similar to295318600792Unknown295418600878Unknown295518600890Unknown295618601250Unknown295718601419Unknown295818601439Unknown295918601460Unknown296018601629huntingtin interacting protein-1-related (H. sapiens), similar to296118601927Unknown296218602066Unknown296318602347Unknown296418602382chromosome condensation-related SMC-associated protein 1296518602858PUTATIVE NUCLEOSIDE DIPHOSPHATE KINASE (NDK) (NDPKINASE), similar to296618602966Unknown296718603033Unknown296818603423Unknown296918603588solute carrier family 1 (glial high affinity glutamate transporter), member 2297018603701Unknown297118603711Unknown297218603795Unknown297318603941PHOSPHATIDYLINOSITOL 3-KINASE REGULATORY SUBUNIT (IB PI3-KINASE P101 SUBUNIT) (PTDINS-3-KINASE P101) (PI3K) (P101-PI3K),similar to297418604379Unknown297518604520Unknown297618604537rab-related GTP-binding protein297718604876exostoses (multiple) 2 (H. sapiens), similar to297818605074Unknown297918605322Unknown298018605359Unknown298118606573Unknown298218645167annexin A2298318676544Unknown298418676570Unknown298518676847Unknown298618860829optic atrophy 1, isoform 1298718860843optic atrophy 1, isoform 7298818916767Unknown298918916841Unknown299018959202leucine-rich PPR-motif containing; leucine-rich protein mRNA299119115954dynein, axonemal, heavy polypeptide 5299219263915Unknown299319353103Unknown299419526647oxidored-nitro domain-containing protein299519584385Unknown299619684029Unknown299719743821integrin beta 1 isoform 1C-2 precursor; integrin VLA-4 beta subunit;fibronectin receptor beta subunit299819850567breast carcinoma amplified sequence 3299919923102holocarboxylase synthetase (biotin-[proprionyl-Coenzyme A-carboxylase(ATP-hydrolysing)] ligase); Holocarbyoxylase synthetase;holocarboxylase synthetase300019923233sterol carrier protein 2300119923611Unknown300219923717rhysin 2300319923721pre-T-cell receptor alpha precursor300419923757golgi autoantigen, golgin subfamily a, 2; golgin-95300520070212voltage-dependent anion channel 3300620070798androgen-regulated short-chain dehydrogenase/reductase 1300720127408hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme Athiolase/enoyl-Coenzyme A hydratase (trifunctional protein), alphasubunit; Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-CoenzymeA thiolase/300820127473glucose regulated protein, 58 kD300920127510peroxisomal long-chain acyl-coA thioesterase; peroxisomal long-chainacyl-coA thioesterase; putative protein301020140018mitochondrial ribosomal protein S9, precursor (MRP-S9)301120140250Sideroflexin 1301220141424Short chain 3-hydroxyacyl-CoA dehydrogenase, mitochondrial precursor(HCDH)301320141538Homeobox protein Hox-C12 (Hox-3F)301420141568Isocitrate dehydrogenase [NADP], mitochondrial precursor(Oxalosuccinate decarboxylase) (IDH) (NADP+-specific ICDH) (IDP)(ICD-M)301520141580Mitochondrial 2-oxoglutarate/malate carrier protein (OGCP)301620141765Succinyl-CoA ligase [GDP-forming] alpha-chain, mitochondrial precursor(Succinyl-CoA synthetase, alpha chain) (SCS-alpha)301720141946DNA topoisomerase II, beta isozyme301820147036transient receptor potential cation channel protein301920150348Deoxy Hbalphayq, A Mutant Of Hba302020151189Glutamate Dehydrogenase-Apo Form302120178093Suppressor of cytokine signaling 7 (SOCS-7) (Nck, Ash andphospholipase C gamma-binding protein) (Nck-associated protein 4)(NAP-4)302220268814CD36 antigen (collagen type I receptor, thrombospondin receptor)302320270305synaptotagmin-like 5302420270399polycystic kidney and hepatic disease 13025226207dihydrolipoamide S-acetyltransferase

[0157] Table 2 presents a selected subset of the 3025 human heart mitochondrial proteins that are disclosed in Table 1 and in the Sequence Listing. The mitochondrial proteins of Table 2 are organized according to particular mitochondrial function classifications as indicated, based on analysis of amino acid sequences and GENBANK annotations; a number of the entries in Table 2 may use earlier GENBANK Accession numbers which differ from those shown in Table 1, but the sequences of such GENBANK Accession numbers can each be matched to a sequence in the Sequence Listing of the instant application using sequence database searching software tools as exemplified above and as known to the art (e.g., Basic Local Alignment Search Tool (“BLAST”), http://www.ncbi.nim.nih.gov/BLAST, Altschul, J. Mol. Biol. 219:555-565, 1991, Henikoff et al., Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992; PSI-BLAST, ALIGN, MEGALIGN; WISETOOLS. CLUSTAL W, Thompson et al., 1994 Nucl. Ac. Res. 22:4673; CAP, www.no.embnet. org/clustalw.html; FASTA/FASTP, Pearson, 1990 Proc. Nat. Acad. Sci. USA 85:2444, available from D. Hudson, Univ. of Virginia, Charlottesville, Va.). As described above, each amino acid sequence provides a polypeptide structure from which a sample can be analyzed to determine, on the basis of structure, whether a modified polypeptide as provided herein may be present in the sample. As also described above, each functional classification refers to a defined biological activity measureable according to methods provided herein and known to the art, such that the invention contemplates determination in a sample of whether a polypeptide that exhibits altered biological activity is present.

2TABLE 2MITOCHONDRIAL FUNCTIONS OF SELECTEDCOMPONENTS OF THE HUMAN HEARTMITOCHONDRIAL PROTEOMEMITOCHONDRIAL FUNCTIONGENBANKSEQ IDCLASSIFICATIONACC NO.NO:Amino acid metabolism11853335126958125634504067754758714527662412241154586352012653507761302764049113518228519147644122401477554650616877964453Amino acid metabolism Total12Apoptosis2286145159104371448431083517363712382773158147294751011476149671716163817100Apoptosis Total7C-compound metabolism1354222404758498405112759863601142823037114365333612230075359126529813611363086239140431873814724751695C-compound metabolism Total10Carrier1134631534505775157455740315576573475321114188585112232421920126538275311363261615213647558151147472161541475202485014774525156Carrier Total12Complex 113013599126257958312625805924505355620450535760945053596134505361611450536561745053676054689104610475876860047587726214758776607475878461447587866014758790588475879258648268486124826852608489437061960416696167657369591100926575851017959962210764847618108350255961083508758412005918369130971565981327256760213272568604135289605901363760860614336775623147690516151477731358715307634595Complex 1 Total37Complex 24759080865136391147921472748686716157047791Complex 2 Total4Complex 3117759944117863947190804946135136093492970789331112801923313631678945136496589481473622394214775827943Complex 3 Total10Complex 41171032112262092211262581207450298521345029872184502989217450299121945029932204758038210475804021513629150209136378332161364842623716196598212Complex 4 Total14Complex 5114549841262582804502297874502303935901896896005717881152614985132728558113543618831477413991Complex 5 Total10DNA synthesis1187494971709123281415387484011225260283DNA synthesis Total4Glycolysis31645355107554752129070750136066921387011751455703246711430299401126533716841343641335014043654831147612083561555312740315991827402Glycolysis Total13Guanine-related1061853721210093793867453801335250784450404937845065177646005772747100471183441094542851611055998376147458083771577912637516181084343Guanine-related Total13Inositol-related10848068812450543313991056824505801686108350234311143677843514724557683147282296871476064943214783738434Inositol-related Total10Kinase/phosphatase13074945110367757317092426504503269246450515351045060915514557769522743934673710047120437115267894301264371673812654407574126590077331283036780313606056280136319075531364638522213648611802139386192241419446111147215078011473390479914736227774147403711214749765101478292173214784064552147854057061530148841816033591808Kinase/phosphatase Total30Lipid metabolism10827237221169204286176253314832732281845018692245023279745036072954503609296450365132245049754844557817869455783372447583122971083505931911276083323114330076781164056642112669909483127075703041280502119134353503271363962813136472764651365304920140416993101404345137314725848211472978325214730775420147464878151476415914147642024191476977667414781245324Lipid metabolism Total34Lipoprotein229479480108269269348269146919438229692134700947014721241485Lipoprotein Total6Nucleotide metabolism4502013284502457784503375258867184620413654685791477677877Nucleotide metabolism Total6Protease450220130450256313776569591391004710614412408656136126436372412654627517147726721381478005572716741033726Protease Total10Protein targeting12357138510916883901346317387400813118450321819155802970336912714916765725791776626739189910382533126551953911364549238914603309386Protein targeting Total13ras/GTPase165726678958031357551135987437111436135761126527156481275111770413569962845136512297721365232476013786129417137942677571421157020214249144754147407921390ras/GTPase Total14Receptor1844777711001941257116878131645047334364877291763119681528521363226689413650874748147328868951474423464616161569788Receptor Total11Redox8021506624502601143455784577569125366331139946623911416669632128043191421311202319913236495753135292574113627233421399432574414735899235Redox Total13Stress4503731331475819280054539026347643782383136314406751425006367614755436874Stress Total7Structural1319419745913643253460141249764611473078246215305472924Structural Total5TCA cycle41717845010718342561170477451171850216503177744851745395001132158187211321583868113746644521280490144913627252658136398175051474054734214782063501153188431716192638446TCA cycle Total16Transcription10529448107912905103318214001582692888256503290445064457804507389301667845590869124402879884738671109617178311761696119118907557821265377539412734816741132420696471378719724213938539232147301588891474226678114748858910147663737651479019084715296351859153001495581545185453016163124926Transcription Total27Translation170661130045035073114758118243503205167661872474770562654377063495461117714853511416393538114244045441155992754211596859537130276045471312397673135594045341363152154913648964351389923154114028389539140284055451416527053614285174299151508115481529557446915298022540Translation Total25Transport287145211437457911725541394135971557815882921304503057225572993751857300338487799988470892387040810716563135108352209411612670690128032811395133769911396135406068751364921713931414960718614739472710147677381341477838129416974753849Transport Total22Tumor-related120749498132164768117743812345076439301056716434810835155928108639073971224690192912643796770135290479121365063951514725399898147553369311507682766515296762138816160929769Tumor-related Total16Zinc finger11772301401211702214022317769714302138614034507979140448270651405545418014077671629464142119071410142861861406146703601409147553163025147554561408Zinc finger Total13

Example 4

OXIDATIVE POST-TRANSLATIONAL MODIFICATION OF TRYPTOPHAN RESIDUES IN CARDIAC MITOCHONDRIAL PROTEINS

[0158] This example shows the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid was associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, the susceptible tryptophan residues in this group of proteins appeared, according to non-limiting theory, to be “hot spots” for oxidation in close proximity to a source of reactive oxygen species (ROS) in respiring mitochondria.

[0159] LC/MS/MS data generated from the human heart mitochondrial proteome project as described in the preceding Examples, as well as data for human and bovine proteins prepared by sucrose density gradient centrifugation as described above, or by immunoprecipitation using antibodies against complex V (ATP synthase) and/or complex I (NADH dehydrogenase) proteins (see, Table 2), were queried against the human or bovine subsets of GenBank using the Sonar MSMS searching algorithm (Genomic Solutions, Ann Arbor, Mich.) with oxidation of methionine (+16 u) and tryptophan (+32 u) specified as differential modifications. Corresponding MALDI spectra were manually inspected. FIG. 3 shows oxidation products of tryptophan from proteins, including N-formylkynurenine (Structure 2).

[0160] Modifications to complex I subunits in bovine heart mitochondria in response to the oxidative stress caused by peroxynitrite treatment were studied in vitro, and yielded evidence of oxidized tryptophan in several subunits, both by MALDI TOF and by LC/MS/MS. Surprisingly, the relative intensities of the peaks in the MALDI spectra corresponding to peptides containing N-formylkynurenine were also high in untreated mitochondria from some bovine and human heart preparations, although there was substantial variation. Prior to complex I isolation and electrophoresis, mitochondria were prepared identically from all hearts which were freshly collected, frozen and thawed immediately prior to analysis. FIG. 4 shows the MALDI spectra of peptides from the human complex I subunit, NDUFS4 (see Table 3), and its bovine homologue from five different preparations corresponding to seven different hearts (five human, including one pooled sample of mitochondria from three individual hearts, and two bovine hearts). The relative intensities of m/z 1329.6 and 1361.6 (corresponding to peptides without and with dioxidized tryptophan, FIG. 4A) and 1112.5 and 1128.5 (corresponding to peptides without and with oxidized methionine, FIG. 4B) were used as a rough measure of protein oxidation. No correlation was found between the extent of tryptophan oxidation and that of methionine oxidation, suggesting that they occurred via different mechanisms.

[0161] The dioxidation of tryptophan was clearly discernable in FIG. 4A (i) and (ii) in which complex I was purified by different methods, sucrose density gradient centrifugation or immunoprecipitation, respectively, but corresponded to mitochondria from the same human heart. This finding suggested that the method of preparation was not a factor in determining the extent of oxidation, but rather that such oxidation was a characteristic of the donor from which the sample was obtained (in this case, a 41-year-old male Caucasian who died of brain cancer). The other human donor, displaying far less extensive oxidation of tryptophan as seen in FIG. 4A (iii), was a 62-year-old female Caucasian who died of intracranial bleeding. In contrast, NDUFS4 from a pool of mitochondria from three human hearts displayed an extensively oxidized tryptophan-containing peptide FIG. 4A (iv). Again the degree of oxidation in the pooled sample was not commensurate with the degree of oxidation for the methionine-containing fragment FIG. 4B (iv).

[0162] Distribution of the oxidatively modified tryptophan in the MS/MS spectra dataset described in the preceding Examples was assessed by reanalyzing the data with N-formylkynurenine selected as a differential modification of tryptophan (+32) using the SonarMSMS algorithm according to the supplier's instructions (Genomic Solutions, Ann Arbor, Mich.). Table 3 lists N-formylkynurenine-containing peptides found with peptide expect scores (Epep) values ≦1×10−2 (99% confidence); also listed in Table 3 are the identifiers for the mitochondrial polypeptide sequences from which these peptides derived. Of this list of 51 peptide sequences from 39 proteins, 9 subunits of complex I had N-formylkyenurine-containing tryptic peptides and included two newly discovered subunits (Table 1, NCBI/Genbank Acc. Nos. 13938442 and 17455445, now 21754001). This subset of proteins was used to compare tryptophan oxidation versus methionine oxidation as a function of the ability to observe a peptide in any given LC/MS/MS experiment. As shown in FIG. 5, the numbers of distinct peptides containing methionine (A) and tryptophan (B) were plotted for a given complex I subunit which had a Sonar MSMS Epep score of ≦1×10−2, and on each plot FIG. 5 indicates whether the corresponding oxidized residue was observed. Methionine oxidation appeared to be directly related to the number of observable peptides that would be expected if oxidation were a random sample-handling artifact. In contrast, tryptophan oxidation appeared to be much more specific to selected subunits, with the greatest modification being noted for NDUFV1 (51 kDa flavoprotein 1) and NDUFA9 (a 39 kDa reductase/isomerase subunit). In addition, five subunits of the iron-protein component were oxidized.

3TABLE 3PEPTIDES CONTAINING DOUBLY OXIDIZEDTRYPTOPHAN FROM THE CARDIACMITOCHONDRIAL PROTEOME.PeptideDerivedfromNCBI/GenbankPROTEINPEPTIDEEpepAcc. No.DESCRIPTIONVFEISPFEPwITR1.40E−056681764NDUFA9FGPIPLGSLGwK2.30E−046681764NDUFA9wLSAEIEDVKPAK1.80E−036681764NDUFA9HAGGVTGGwDNLLAVIPGGS2.10E−0420149568NDUFV1STPLIPKGDARPAEIDSLwEISK9.40E−0420149568NDUFV1GPDwILGEIK2.40E−0320149568NDUFV1LAALPENPPAIDwAYYK3.20E−055453559ATPase d F0TIDwVAFAEIIPQNQK2.10E−035453559ATPase d F0YPYwPHQPIENL7.20E−035453559ATPase d F0wVVIGDENYGEGSSR8.40E−083600098aconitase precursorVAEKEGwPLDIR4.00E−043600098aconitase precursorLwISNGGLADIFTVFAK2.90E−0618044943acyl-Coenzyme Adehydrogenase, very long chainIFGSEAAwK3.90E−0318044943acyl-Coenzyme Adehydrogenase, verylong chainALGVLAQLIwSR1.10E−054758076citrate synthase precursorDYIwNTLNSGR7.10E−044758076citrate synthaseprecursorKLETAVNLAwTAGNSNTR1.60E−054507879VDAC-1wNTDNTLGTEITVEDQLAR5.30E−034507879VDAC-1VVDGAVGAQwLAEFR4.70E−0517458911dihydrolipoamideS-acetyltransferaseVPEANSSwMDTVIR6.60E−0417458911dihydrolipoamideS-acetyltransferaseSAVTALwGK3.70E−034504349beta globinLLVVYPwTQR4.30E−034504349beta-globinRPPEPTTPwQEDPEPEDENL6.80E−0813938442neuronal proteinYEK(ND17.3)NLTQYSwLLDGFPR1.00E−0619923437adenylate kinase 3alpha likeFDLNSPwEAFPVYR2.10E−0511360206NDUFS3IASGLGLAwIVGR2.60E−054758714microsomal glutathioneS-transferase 3GYIVIEDLwK2.90E−0512001992brain my025ASSTSPVEISEwLDQK4.00E−054503607electron transferflavoprotein alphapolypeptideGRPTSTNPIASIFAwTR6.40E−054504575isocitratedehydrogenase 2(NADP+),mitochondrialGLLTYTSwEDALSR1.40E−0421411235NDUFS1IPwFQYPIIYDIR1.90E−046005854D-prohibitinGLSDGEwQLVLNVwGK2.50E−04229361MyoglobinASwSSLSMDEK3.00E−045921895Cytochrome c oxidasesubunit IV isoform 1LDDLVNwAR5.30E−0421750696NDUFS7TLLwTELFR7.80E−044505371NDUFS8SYGANFSwNK8.70E−0413528960NDUFS4ASLHALVGSPIIwGGEPR9.90E−0413676336long-chain acyl-coAthioesteraseperoxisomalwEVADLQPQLK1.20E−0321903482Ubiquinol-cytochromeC reductase complexcore protein 2YEGFFSLwK1.30E−0321361114mitochondrial carrier;oxoglutarate carrierLITTQQwLIK1.40E−0313272660ATP synthase 6LWEPLVEEPPADQwK1.50E−034826848NDUFA5IDEAILITwTK2.00E−0315991833hexokinase 1wDGQETTLVR3.30E−03458862fatty acid bindingprotein, heart; hFABPHwLDSPwPGFFTLDGQPR3.40E−03205415922-oxoglutaratedehydroqenase E1component,mitochondrialprecursorAwNGSAEGPGKVER4.30E−0321754001Unnamed proteinproduct (NDUFB11)ELwFSDDPNVTK4.70E−034757732programmed cell death8 (apoptosis-inducingfactor AIF)EQwDTIEELIR5.30E−0345033012,4-dienoyl CoAreductase 1 precursorGAwSNVLR5.30E−0386754carrier ANTwYYNAAGFNK5.30E−035454152UCR ubiquinone-binding protein (VI)ELDSITPEVLPGwK5.50E−038131894MitofilinAPLAEEwDNMTMK8.10E−034505093monoamine oxidase BLATFwYYAK9.10E−0322096328ATP synthase G chain,mitochondrial

[0163] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Number	Date	Country
60412418	Sep 2002	US
60389987	Jun 2002	US
60372843	Apr 2002	US

Targets for therapeutic intervention identified in the mitochondrial proteome

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