Yeast proteome analysis

FIELD OF THE INVENTION

[0002] The invention relates to high-throughput proteome analysis.

BACKGROUND OF THE INVENTION

[0003] Cellular behavior is determined by the dynamic interactions of a vast array of proteins that form complexes and higher order networks1. The global coordination of cellular function is presumed to require the concerted regulation of such networks. As the human genome is predicted to contain more than 30,000 discrete open reading frames, which may each give rise to multiple protein variants via splicing and other modifications, the problem of systematically decoding protein interactions is daunting. To date, attempts to generate comprehensive protein-protein interaction maps have relied on the yeast two-hybrid system, whereby binary interactions are detected via bridging of transcription factor DNA binding and transactivation domains, thereby activating reporter gene expression2. Large scale applications of the two-hybrid method have yielded numerous relevant protein-protein interactions3-5. In a more direct approach, protein complexes can be purified from cell lysates followed by identification of each constituent. With the advent of ultra-sensitive mass spectrometric protein identification methods, it has become feasible to consider such an approach on a proteome-wide scale6-8.

SUMMARY OF THE INVENTION

[0004] The instant invention is related to the high-throughput (HTP) analysis of protein interaction networks by highly sensitive mass spectrometric identification methods (HTP-MS/MS), also known as high throughput MS/MS protein complex identification (HMS-PCI).

[0005] One aspect of the invention provides a method of identifying a protein interaction network using high throughput tandem mass spectrometry, particularly in the setting of proteome-wide analysis. Typically, a bait protein (either in its native form or a modified form—such as an epitope tagged form) is used to retrieve binding prey proteins from an environment, preferably a native environment inside a cell, and complexes comprising the bait and prey proteins are separated and subjected to mass spectrometry analysis to identify prey proteins.

[0006] Thus in one aspect, the invention provides a method for identifying a protein interaction network comprising two or more bait proteins, comprising: (a) isolating complexes comprising at least one of said two or more bait proteins and their prey proteins from a sample; (b) separating said complexes; and (c) determining the identity of the prey proteins in each of said complexes using mass spectrometry, thereby identifying the protein interaction network.

[0007] In another aspect, the invention provides a method for identifying a protein interaction network comprising two or more bait proteins, comprising: (a) contacting said two or more bait proteins with a sample containing potential prey proteins, wherein the bait proteins and complexes comprising at least one said bait protein(s) are capable of being separated from other proteins in the sample; (b) separating said complexes comprising at least one said bait proteins and their prey proteins; (c) identifying prey proteins in the complexes using mass spectrometry, thereby identifying the protein interaction network.

[0008] In one embodiment, the protein interaction network comprises 5, 10, 20, 50, 100, 200 or more bait proteins. In a related embodiment, the protein interaction network comprises 2%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90%, or 100% of the proteome of a given genome. In a preferred embodiment, the proteome is a yeast (such as S. cerevisiae or S. pombe) proteome.

[0009] In another embodiment, the protein interaction network comprises all bait proteins known to be involved in the same biochemical pathway or biological process.

[0010] In another embodiment, the protein interaction network comprises the same type of proteins, for example, protein kinases, protein phosphatases, receptors, G proteins, ion channels, transcription factors, etc.

[0011] In one embodiment, a bait protein or protein of interest used in a method of the invention is unmodified. In another embodiment, a bait protein or protein of interest is synthesized as a fusion protein with a heterologous polypeptide to facilitate its retrieval from said biological sample. Examples of the heterologous polypeptides include: GST, HA epitope, c-myc epitope, 6-His tag, FLAG tag, biotin, or MBP. Bait proteins can be expressed in a host cell as an exogenous polypeptide.

[0012] A bait protein may be immobilized to facilitate isolation of the complexes. For example, a bait protein may be directly or indirectly (e.g. with an antibody specific for the epitope tag) bound to a suitable carrier or solid support such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.

[0013] In a preferred embodiment, the sample is a biological sample, preferably an extract of a cell. In one embodiment, the extract is concentrated. The cell can be a yeast cell, or it can be a higher eukaryotic cell, such as a nematode (C. elegans), insect, fish, reptile, amphibian, plant, or mammalian cell, or more preferably, a human cell.

[0014] In one embodiment of the invention, complex formation between bait and prey proteins is induced using an extracellular or intracellular factor.

[0015] In one embodiment, complexes comprising at least one bait protein and its prey proteins are isolated by immunoprecipitation. In a related embodiment, complexes are isolated by a GST pull-down assay.

[0016] In one embodiment, complexes are digested by protease before separation. The digestion can be performed on either purified protein or on protein samples in gel.

[0017] In one embodiment, complexes are separated by SDS-PAGE. In a related embodiment, complexes are separated by chromatography, such as HPLC, or any other suitable protein separation means commonly known in the art, including chromatography, HPLC, Capillary Electrophoresis (CE), isoelectric focusing (IEF).

[0018] In a particular embodiment, complexes are separated by SDS-PAGE, and digested by in-gel protease digestion.

[0019] In an aspect, the mass spectrometry employed in a method of the invention is tandem mass spectrometry (MS/MS). In a preferred embodiment, the MS/MS is coupled with Liquid Chromatography (LC).

[0020] In another embodiment, protein sequences obtained from tandem mass spectrometry are compared against protein sequence databases in order to determine the identity of the proteins. In a preferred embodiment, said protein sequence databases include a combination of public database and proprietary database. For example, computer programs including but not limited to the following may be used: TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Altschul et al., 1990, J. Mol. Biol. 215(3):403-10; see, Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-8; Thompson, et al., 1994, Nucleic Acids Res. 22(22):4673-80; Higgins, et al., 1996, Methods Enzymol 266:383-402).

[0021] In another embodiment, the method further comprises repeating steps (a)-(c) using prey proteins identified from previous round(s) as new bait proteins, wherein said new bait proteins are different from any bait proteins used in said previous round.

[0022] The invention also provides libraries of information on a protein interaction network identified using a method of the invention, methods to construct such libraries, and data sharing systems which enable efficient utilization of such libraries. Furthermore, the invention provides databases which accommodate and maintain libraries of information relative to such protein interaction network, methods and systems to construct such databases, methods and systems to enable a user/client to search through such databases for desired information, methods and systems to transmit to a client desired pieces of information concerning protein interaction networks that are housed in databases, tangible electronic means to record and make use of such systems and databases, and apparatus to enable construction and search of databases and/or transmission of desired information to a client. Detailed methods of creating databases as described herein and search engines for these databases, based on information obtained using a suitable method of the invention, are well-known in the art, and thus will not be described in detail.

[0023] Therefore, in one aspect, the invention provides a database of protein interaction network(s) identified by a method of the instant invention, comprising information regarding two or more bait proteins and their interactions.

[0024] In one embodiment, the information includes: the identity of all bait proteins and their interacting prey proteins, the conditions under which the interactions are observed and/or the identity of the sample from which said information is obtained.

[0025] In one embodiment, one or more filters are used to modify the protein interaction network database.

[0026] In one embodiment, the database is verified by information obtained from a public or proprietary database.

[0027] In one embodiment, the database comprises a set of potential protein interactions and molecular complexes in a given proteome, under one or more specific conditions. In a related embodiment, the database comprises at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the potential protein interactions of a given organism The database can also include annotations of certain protein-protein interaction information obtained from searching available scientific literature using proprietary software. Such annotations can be dynamically updated, preferably automatically, by repeated searches performed at predetermined time intervals.

[0028] In one embodiment, the database comprises a set of protein interactions, preferably a set of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the protein interactions, in a yeast cell. In a related embodiment, the database comprises all homologous proteins related to any given set of yeast protein interactions. “Homologous” as used herein means any protein that is at least 75%, preferably 80%, 85%, 90%, or most preferably 95%, even 99% identical to a given protein. Usually, a homologous protein exists in a different species, such as in a worm, insect, plant, or mammal, most preferably in human.

[0029] In one aspect of the invention, a database is provided comprising a yeast protein interaction network. In a particular embodiment, the database comprises a set of more than 4000 yeast protein interactions. In another particular embodiment, the database comprises about 20-30%, preferably about 25-30%, more preferably about 29% of the yeast proteome. In a preferred embodiment, the database comprises the complexes of Table 2, 4A, 4B, 5A, 5B, and 7.

[0030] Another aspect of the invention provides a method of identifying differences in protein interaction networks comprising one or more selected bait proteins, comprising:

[0031] (a) providing a first protein interaction network identified by (i) isolating complexes comprising a selected bait protein(s) and prey proteins from a first sample; (ii) separating complexes comprising the bait protein(s) and prey proteins; and (iii) determining the identity of the prey proteins, preferably by mass spectrometry, thereby identifying the first protein interaction network;

[0032] (b) providing a second protein interaction network identified by (i) isolating complexes comprising the selected bait protein(s) and prey proteins from a second sample; (ii) separating complexes comprising the bait protein(s) and prey proteins; and (iii) determining the identity of the prey proteins, preferably by mass spectrometry, thereby identifying the second protein interaction network; and

[0033] (c) comparing the first and second protein interaction networks, thereby identifying differences in the protein interaction networks.

[0034] In one embodiment, the first sample is from a tumor tissue, and the second sample is from a normal tissue of the same tissue type. In another embodiment, the tumor tissue and the normal tissue are from the same patient. In another embodiment, the first sample and the second sample are from different developmental stages of the same organism. In another embodiment, the first sample is from a tissue, and the second sample is from the same tissue type after a treatment. Such tissue can be, for example, a tumor tissue. Such treatment can be, for example, chemotherapy or radiotherapy.

[0035] The invention also provides methods for assaying for changes in protein interaction networks in response to intracellular or extracellular factors.

[0036] Therefore, a method is provided for assaying for changes in protein interaction networks in response to an intracellular or extracellular factor comprising: (a) contacting two or more bait proteins with a sample containing prey proteins in the presence of an intracellular or extracellular factor, wherein the bait proteins and complexes comprising the bait proteins are capable of being separated from other proteins in the sample; (b) separating complexes comprising bait proteins and prey proteins; (c) identifying prey proteins in the complexes using mass spectrometry, thereby identifying the protein interaction network; and (d) comparing the protein interaction network identified in (c) with a protein interaction network identified in the absence of the intracellular or extracellular factor.

[0037] Another aspect of the invention provides a method to identify potential protein targets for drug design and pharmaceutical research, comprising identifying a network of protein interactions comprising a protein of interest, such as a previously known drug target, using the method or database of the instant invention, thereby identifying other related drug targets for a given biological process.

[0038] Thus, in this respect, the invention provides a method of conducting a pharmaceutical business, comprising: (a) identifying a protein interaction network of one or more known bait protein from a sample using a method of the invention wherein said bait protein is a potential drug target; (b) identifying, among prey proteins that interact with said bait protein in the protein interaction network, new potential drug targets; (c) licensing, to a third party, the rights for further drug development of inhibitors or activators of the drug target.

[0039] In a related aspect, the invention provides a method of conducting a pharmaceutical business, comprising: (a) identifying a protein interaction network of one or more known bait proteins from a biological sample using a method of the invention, wherein said bait protein is a potential drug target; (b) identifying, among prey proteins that interact with said bait proteins in the protein interaction network, new potential drug targets; (c) identifying compounds that modulate activity of said new potential drug targets; (d) conducting therapeutic profiling of compounds identified in step (c), or further analogs thereof, for efficacy and toxicity in animals; and, (e) formulating a pharmaceutical preparation including one or more compounds identified in step (d) as having an acceptable therapeutic profile.

[0040] In one embodiment, the method further comprises an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale. In a related embodiment, the method further comprises establishing a sales group for marketing the pharmaceutical preparation.

[0041] Methods and reagents provided by the instant invention are useful for rapid, efficient identification of protein-protein interactions in a large scale. In one respect, it provides a platform for doing drug screen related pharmaceutical research in a genetically well defined system such as yeast, by virtue of sequence homology between yeast and its higher eukaryotic counterparts such as human. In another respect, it also offers a high throughput means to study protein-protein interaction and signaling networks directly in higher organisms. The ultimate utility of any large scale platform rests upon its ability to reliably glean new insights into biological function. By the criterion of extensive literature validation, initial study demonstrates that the HTP-MS/MS approach is well suited to this task Given that the encoded set of human proteins is nominally 5-fold greater than the set of predicted yeast proteins, comprehensive analysis of the human proteome is feasible with current HTP-MS/MS platforms.

[0042] The methods of the present invention, as described above, may be practiced using kits for identifying protein interaction networks comprising two or more bait proteins. A kit will generally include expressable recombinant vectors for generating bait proteins.

[0043] The invention also provides a method for constructing a protein interaction network map for a proteome comprising: (a) identifying a protein interaction network using a method of the invention, and (b) displaying the network as a linkage map.

[0044] The invention also provides an integrated modular system for performing methods of the invention. In an embodiment, the system comprises one or more of the following modules: (a) a module for retrieving recombinant clones encoding bait proteins; (b) an automated immunoprecipitation module for purification of complexes comprising bait and prey proteins; (c) an analysis module for further purifying the proteins from (b) or preparing fragments of such proteins that are suitable for mass spectrometry; (d) a mass spectrometer module for automated analysis of fragments from (c); (d) a computer module comprising an integration software for communication among the modules of the system and integrating operations; and (e) a module for performing an automated method of the invention.

[0045] The integrated modular system may be automated for high throughput operation.

[0046] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization B. D. Hames & S. J. Higgins eds. (1985); Transcription and Translation B. D. Hames & S. J. Higgins eds (1984); Animal Cell Culture R. I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984).

DESCRIPTION OF DRAWINGS

[0047] The invention will be better understood with reference to the drawings in which:

[0048]
FIG. 1 illustrates a HMS-PCI strategy a, Flow diagram of approach b, Protein complexes captured onto anti-FLAG agarose resin, eluted and resolved by SDS-PAGE c, Proteins specific to the elution are excised, digested with trypsin and subject to LC-MS/MS. Matches of fragmentation spectra to databases unambiguously identify proteins in the sample, as shown here for Ste12.

[0049]
FIG. 2 illustrates kinase-based signaling networks a, The mating pheromone MAPK pathway. The core Ste11-Ste7-Fus3-Kss1 MAPK module phosphorylates downstream transcription factors and other targets. Blue indicates proteins identified in association with Kss1 b, Interaction diagram for Kss1 complexes c, Interaction diagram for Cdc28 complexes. Arrows point from the bait protein to the interaction partner. Black arrows indicate known interactions; red arrows indicate novel interactions.

[0050]
FIG. 3 illustrates the DNA damage response network. Interactions were initially nucleated from 86 proteins implicated in the DDR. Blue nodes indicate known interactions within dedicated complexes as labeled. Black arrows indicate known interactions; red arrows indicate novel interactions.

[0051]
FIG. 4 shows a graphical representation of large-scale protein interaction networks and comparison to literature interactions a, entire HMS-PCI network in spoke model representation b, overlap of spoke model and PreBIND c, overlap of HTP-Y2H dataset3 and PreBIND d, overlap of spoke model and HTP-Y2H dataset3. Blue nodes and edges are literature-derived interactions; red nodes and edges are novel interactions detected by HTP approaches. For clarity, simple binary interactions are not shown in panels b (36 interactions removed), c (20 interactions removed) and d (30 interactions removed).

[0052]
FIG. 5 shows the percentage of total baits bound per each interacting protein. Each interacting protein was plotted versus the percentage of the total baits it bound. To the left of the dotted line, the percentage of total baits bound increases dramatically. This corresponds to 3% of total baits bound, and was taken as the percentage of baits bound that at and above which the interacting protein is likely a background, promiscuous binder.

DETAILED DESCRIPTION OF THE INVENTION

[0053] Definitions

[0054] “Binding,” “bind” or “bound” refers to an association, which may be a stable association, between two molecules, e.g., between a protein ligand and a another polypeptide, due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions.

[0055] “Bait” or “bait protein” refers to proteins used in an assay aimed at identifying interacting or “prey” proteins to preferably define a protein interaction network. A bait protein may comprise all or part of a target molecule which has been implicated in a biological process of interest, or for which the function is sought. A bait protein may include functional domains of a wide variety of proteins including receptors, ligands, enzymes, transcription proteins, cell cycle proteins, etc. In an aspect of the invention, bait proteins are selected from a proteome (e.g. yeast) including but not limited to yeast proteins implicated in DNA damage and repair, protein kinases, protein phosphatases, receptors, G proteins, ion channels, and transcription factors.

[0056] A bait protein may be in its native form, or may be modified to facilitate the identification process. For example, the bait protein may be synthesized as a fusion protein so that it contains a heterologous domain/motif that is useful for isolating the fusion protein. Any known or commonly used polypeptides for which an isolation method is available can be utilized as the heterologous domain in the bait fusion protein. Such heterologous domains may include (but are not limited to) GST, an epitope tag (FLAG tag, c-myc tag, HA (human Influenza virus hemagglutinin) tag, or other commonly used or commercially available epitope tags, etc.), 6-His tag, biotin, GFP (green fluorescent protein), MBP (Maltose Binding Protein), etc. An advantage of using the fusion bait protein is that the need to prepare an antibody for each potential bait protein is obviated, and relatively uniform efficiency of retrieving complexes containing the bait proteins can be achieved. Also, the fusion protein may be easily differentiated from the endogenous proteins, which may or may not be expressed in a given cell at a given time.

[0057] “Prey” or “prey protein” refers to any polypeptide that binds to a “bait” protein, either directly by binding to the bait protein, or indirectly by binding to other proteins so that the bait and the prey exist in the same multi-polypeptide complex, under a given condition, including a native or physiological condition or an experimental condition.

[0058] “Complex” generally refers to an association between at least two moieties (e.g. chemical or biochemical) that have an affinity for one another. Examples of complexes include associations between antigen/antibodies, lectin/avidin, antibody/anti-antibody, receptor/ligand, enzyme/ligand and the like. “Member of a complex” refers to one moiety of the complex, such as an antigen or ligand, or a bait and a prey. “Protein complex” or “polypeptide complex” refers to a complex comprising at least one polypeptide. In the context of the present invention, a complex includes a prey protein bound to a bait protein.

[0059] “Exogenous” means caused by factors or an agent from outside the organism or system, or introduced from outside the organism or system, specifically: not normally synthesized within the organism or system. A fusion/tagged protein expressed from an introduced plasmid may be considered exogenous to the host cell expressing the fusion protein, although the host itself may express an endogenous version of the same protein.

[0060] “Extracellular factor” includes a molecule or a change in the environment that is transduced intracellularly via cell surface proteins (e.g. cell surface receptors) that interact, directly or indirectly, with a signal. An extracellular factor includes any compound or substance that in some manner specifically alters the activity of a cell surface protein. Examples of such signals or factors include, but are not limited to growth factors, that bind to cell surfaces and/or intracellular receptors and ion channels and modulate the activity of such receptors and channels. The signals and factors include analogs, derivatives, mutants, and modulators of such growth factors.

[0061] “Intracellular factor” includes a molecule or a change in the cell environment that is transduced in the cell via cytoplasmic proteins that interact, directly or indirectly with a signal. An intracellular factor includes any compound or substance that in some manner specifically alters the activity of a cytoplasmic protein involved in a biological or signal transduction pathway.

[0062] “Filter” when referring to data processing means eliminating certain obtained/observed data based on certain preset criteria For example, a protein sample loaded onto one lane of a SDS-PAGE gel may occasionally spill-over the adjacent lanes, which may be subsequently detected by the highly sensitive MS/MS analysis. Thus, a protein that is the same as a bait protein on gel loaded within 3 gel lanes on either side of the bait protein on a gel may be designated as a “spillover,” and filtered from the data set. More than one filter set can be used to modify the final protein interaction network.

[0063] “GST pull-down assay” refers to a method comprising incubating GST-fusion proteins within a sample (such as cell lysate) with GST-binding moieties, typically glutathione beads, and “pulling-down,” proteins binding to the GST-fusion protein. The process is analogous to immunoprecipitation using antibodies against specific proteins.

[0064] “High throughput” refers to the ability to process large amount of samples in a given process, method, or assay, etc. In a preferred embodiment, the high throughput process is conducted with an automated machine(s), which is optionally controlled by computer software or human or both.

[0065] “Hit” generally refers to a desired result in an assay. For example, in an assay searching for interacting proteins of a given “bait” protein, a hit refers to a “prey” protein that is identified by the assay/process as being able to interacting with the bait protein.

[0066] “Molecular complex” refers to assemblages composed of more than two polypeptides. Each component of the molecular complex binds together by non-covalent bonds. There is no limitation on the number of proteins of the complex. Preferably, a molecular complex comprises two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, or thirty interacting proteins that potentially have a common origin, function, structure, mechanism, or activity.

[0067] “Analyzing a protein by mass spectrometry” or similar wording refers to using mass spectrometry to generate information which may be used to identify or aid in identifying a protein. Such information includes, for example, the mass or molecular weight of a protein, the amino acid sequence of a protein or protein fragment, a peptide map of a protein, and the purity or quantity of a protein.

[0068] “Protein interaction network” refers to a collection of information regarding protein-protein interactions among certain proteins. A protein interaction network may contain a number of bait proteins, as well as prey proteins identified as being able to directly or indirectly bind with these bait proteins. A given protein interaction network may be verified and/or expanded by including some of the initially identified prey proteins as bait proteins for subsequent rounds of assays aimed at identifying more interaction proteins. The protein interaction network may be represented using a number of models, for example, see the spoke model and the matrix model described below. A protein interaction network may also be associated with a given condition (cell type, developmental stage, cell-cycle stage, complex isolation condition, etc.) when necessary, since the same set of bait proteins may yield different protein interaction networks under different conditions. Thus a protein interaction network may represent all possible interactions among conditions, or represent interactions observed in a specific condition. A protein interaction network may represent the entire interaction map of a proteome that specifies the entire signal transduction and metabolic networks of a cell such as a yeast cell.

[0069] A protein interaction network typically comprises two or more proteins. In certain protein interaction networks, any two proteins within the network are directly or indirectly connected. In the latter case, if protein A and X are indirectly connected, it includes the situation that protein A binds protein B, and protein X binds protein Y, wherein A and X do not directly interact with each other, but B and Y directly interact with each other, although the A-B, B-Y, and X-Y interactions need not occur under the same condition or in the same sample. It also includes the situation wherein B and Y are indirectly connected via other proteins. This is analogous to the internet wherein any two computers on the internet can be directly or indirectly connected. In certain other protein interaction networks, at least two proteins are not connected to each other, either directly or indirectly. This is analogous to two or more separate local area networks wherein each member of a local area network is only directly or indirectly connected with other members of the same network, but not members belonging to other local area networks.

[0070] “Promiscuous binder” refers to proteins that bind to numerous bait proteins, and which are excluded from a protein interaction network data set.

[0071] “Proteome” refers to all the proteins that can be encoded by a given genome, which is in turn all the genetic material (including all the genes) of a given organism. Not all proteins within a given proteome are necessarily expressed at the same time, in the same cell type/tissue origin. Due to changes in conditions such as developmental, environmental, physiological, or pathological conditions, any given tissue/cell type may only express a fraction of the total number of proteins that can be encoded by a given genome (or, a fraction of the total proteome). “Troteome” may also refer to the entire complement of proteins expressed by a given tissue or cell type.

[0072] “Solid support” or “carrier,” used interchangeably, refers to a material which is an insoluble matrix, and may (optionally) have a rigid or semi-rigid surface. Such materials may take the form of small beads, pellets, disks, chips, dishes, multi-well plates, wafers or the like, although other forms may be used. In some embodiments, at least one surface of the substrate will be substantially flat.

[0073] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules, with identity being a more strict comparison. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of homology or similarity or identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the—sequences of the present invention.

[0074] The term “percent identical” refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md. In one embodiment, the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.

[0075] Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Preferably, an alignment program that permits gaps in the sequence is utilized to align the sequences. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. An alternative search strategy uses MPSRCH software, which runs on a MASPAR computer. MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both polypeptide and DNA databases.

[0076] Databases with individual sequences are described in Methods in Enzymology, ed. Doolittle, supra. Some exemplary public databases include GenBank, EMBL, DNA Database of Japan (DDBJ), SwissProt, PIR and other databases derived therefrom. In comparing a new nucleic acid with known sequences, several alignment tools are available. Examples include PileUp, which creates a multiple sequence alignment, and is described in Feng et al., J. Mol. Evol. (1987) 25:351-360. Another method, GAP, uses the alignment method of Needleman et al., J. Mol. Biol. (1970) 48:443-453. GAP is best suited for global alignment of sequences. A third method, BestFit, functions by inserting gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman, Adv. Appl. Math. (1981) 2:482-489. Alternatively, certain commercial software packages such as LaserGene from DNAStar inc. can be used for certain aspects of sequence analysis. Multiple softwares and databases may be used in any analysis.

[0077] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a natural or recombinant gene product of fragment thereof.

[0078] The term “recombinant protein” refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous polypeptide. Moreover, the phrase “derived from”, with respect to a recombinant gene, is meant to include within the meaning of “recombinant protein” those polypeptides having an amino acid sequence of a native polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the polypeptide.

[0079] Genetic techniques, which allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo, are known to those skilled in the art. For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination of a target sequence. As used herein, the phrase “target sequence” refers to a nucleotide sequence that is genetically recombined by a recombinase. The target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity. Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject target gene polypeptides. For example, excision of a target sequence which interferes with the expression of a recombinant target gene, such as one which encodes an antagonistic homolog or an antisense transcript, can be designed to activate expression of that gene. This interference with expression of the polypeptide can result from a variety of mechanisms, such as spatial separation of the target gene from the promoter element or an internal stop codon. Moreover, the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3′ to 5′ orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5′ end of the coding sequence in an orientation with respect to the promoter element which allows for promoter driven transcriptional activation.

[0080] “Phospho-protein” is meant a polypeptide that can be potentially phosphorylated on at least one residue, which can be either tyrosine or serine or threonine or any combination of the three. Phosphorylation can occur constitutively or be induced.

[0081] “Post-translational modification” is meant any changes/modifications that can be made to the native polypeptide sequence after its initial translation. It includes, but are not limited to, phosphorylation/dephosphorylation, prenylation, myristoylation, palmitoylation, limited digestion, irreversible conformation change, methylation, acetylation, modification to amino acid side chains or the amino terminus, and changes in oxidation, disulfide-bond formation, etc.

[0082] “Sample” as used herein generally refers to a type of source or a state of a source, for example, a given cell type or tissue. The state of a source may be modified by certain treatments, such as by contacting the source with a chemical compound, before the source is used in the methods of the invention. It should be noted that protein interaction network data based on “a sample” does not necessarily comprise results obtained from a single experiment. Rather, to completely determine a protein interaction network, multiple experiments are often needed, and the combined results of which are used to construct the protein interaction network data for that particular sample.

[0083] Methods of the Invention

[0084] A bait protein for use in the methods of the invention can be expressed in high levels in any given host cell using proper molecular biology techniques. A skilled artisan shall be able to determine the best suitable system including expression vectors, suitable host cells, means to introduce heterologous DNA into such host cells, optimal conditions for protein expression, etc. for any given protein. The example herein is provided for illustration purpose only and shall not be construed as a limitation of the scope of the invention in any way.

[0085] A typical vector suitable for host cell expression shall contain at least the necessary elements for transcription and translation of the target protein. To avoid potential toxicity of heterologous protein expression in the host cell, the expression can be under the control of an inducible promoter, such as a galactose-inducible promoter. The vector used can optionally contain an epitope tag against which an antibody, preferably a commercial antibody is available so that the synthesized fusion protein can be readily isolated using a standardized immunoprecipitation procedure.

[0086] To facilitate large scale high throughput experiments, the vector can be further adapted to be compatible with the Gateway™ system (Invitrogen) by including att sites so that batch cloning can be achieved using recombination-based cloning. PCR amplification can then be used to generate gene fragments flanked by att sites for efficient cloning into the Gateway vector. It should be noted that other similar systems of recombination-based cloning can also be used and are also within the scope of the instant invention.

[0087] Generally, any given protein of interest or bait protein can be expressed in a host cell, either with or without an epitope tag against which an antibody is available, and protein complexes encompassing this protein of interest are isolated using any of many suitable techniques such as immunoprecipitation. The isolated complexes can be separated on SDS-PAGE gel and each band representing at least one potentially interacting protein can be digested by protease such as trypsin or other equivalent enzymes that generates C-terminal basic amino acids such as Arg or Lys. The digested protein samples are then analyzed by tandem mass spectrometry (MS/MS) to obtain sequence information of at least a few peptide fragments. These data will then be compared with known sequences in the publicly available protein/polynucleotide database to unequivocally identify those interacting proteins.

[0088] One aspect of the instant invention discloses a method for large scale analysis of protein-protein interactions using ultra-sensitive mass spectrometry. The mass spectrometry platform is based on a high throughput LC-MS/MS approach for protein complex identification, which is referred to herein as HMS-PCI. This platform is much more powerful than commonly used MALDI-TOF platforms. Although MALDI-TOF is capable of high throughput, it does not readily allow for peptide fragmentation and is therefore limited to highly purified preparations from organisms with small genomes. In contrast, LC-MS/MS instrumentation allows identifications to be made from complex protein mixtures because peptide sequence information is obtained. A direct comparison between studies in yeast with a MALDI-TOF instrument and studies on the same samples shows that the LC-MS/MS approach yielded a much greater hit rate. It is worth noting that the HMS-PCI approach is well suited to analysis of complex proteomes (e.g., the human proteome), whereas MALDI-based platforms are not.

[0089] Mass Spectrometers, Detection Methods and Sequence Analysis

[0090] In certain embodiments, the interacting proteins are identified by protease digestion followed by mass spectrometry. During the past decade, new techniques in mass spectrometry have made it possible to accurately measure with high sensitivity the molecular weight of peptides and intact proteins. These techniques have made it much easier to obtain accurate peptide masses of a protein for use in databases searches. Mass spectrometry provides a method, of protein identification that is both very sensitive (10 fmol-1 pmol) and very rapid when used in conjunction with sequence databases. Advances in protein and DNA sequencing technology are resulting in an exponential increase in the number of protein sequences available in databases. As the size of DNA and protein sequence databases grows, protein identification by correlative peptide mass matching has become an increasingly powerful method to identify and characterize proteins.

[0091] Mass Spectrometry

[0092] Mass spectrometry, also called mass spectroscopy, is an instrumental approach that allows for the gas phase generation of ions as well as their separation and detection. The five basic parts of any mass spectrometer include: a vacuum system; a sample introduction device; an ionization source; a mass analyzer; and an ion detector. A mass spectrometer determines the molecular weight of chemical compounds by ionizing, separating, and measuring molecular ions according to their mass-to-charge ratio (m/z). The ions are generated in the ionization source by inducing either the loss or the gain of a charge (e.g. electron ejection, protonation, or deprotonation). Once the ions are formed in the gas phase they can be electrostatically directed into a mass analyzer, separated according to mass and finally detected. The result of ionization, ion separation, and detection is a mass spectrum that can provide molecular weight or even structural information.

[0093] A common requirement of all mass spectrometers is a vacuum. A vacuum is necessary to permit ions to reach the detector without colliding with other gaseous molecules. Such collisions would reduce the resolution and sensitivity of the instrument by increasing the kinetic energy distribution of the ion's inducing fragmentation, or preventing the ions from reaching the detector. In general, maintaining a high vacuum is crucial to obtaining high quality spectra.

[0094] The sample inlet is the interface between the sample and the mass spectrometer. One approach to introducing sample is by placing a sample on a probe which is then inserted, usually through a vacuum lock, into the ionization region of the mass spectrometer. The sample can then be heated to facilitate thermal desorption or undergo any number of high-energy desorption processes used to achieve vaporization and ionization.

[0095] Capillary infusion is often used in sample introduction because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a mass spectrometer with other separation techniques including gas chromatography (GC) and liquid chromatography (LC). Gas chromatography and liquid chromatography can serve to separate a solution into its different components prior to mass analysis. Prior to the 1980's, interfacing liquid chromatography with the available ionization techniques was unsuitable because of the low sample concentrations and relatively high flow rates of liquid chromatography. However, new ionization techniques such as electrospray were developed that now allow LC/MS to be routinely performed. One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/preparation and mass spectrometer analysis.

[0096] In terms of sample ionization, two of the most recent techniques developed in the mid 1980's have had a significant impact on the capabilities of Mass Spectrometry: Electrospray Ionization (ESI) and Matrix Assisted Laser Desorption/Ionization (MALDI). ESI is the production of highly charged droplets which are treated with dry gas or heat to facilitate evaporation leaving the ions in the gas phase. MALDI uses a laser to desorb sample molecules from a solid or liquid matrix containing a highly UV-absorbing substance.

[0097] The MALDI-MS technique is based on the discovery in the late 1980s that an analyte consisting of, for example, large nonvolatile molecules such as proteins, embedded in a solid or crystalline “matrix” of laser light-absorbing molecules can be desorbed by laser irradiation and ionized from the solid phase into the gaseous or vapor phase, and accelerated as intact molecular ions towards a detector of a mass spectrometer. The “matrix” is typically a small organic acid mixed in solution with the analyte in a 10,000:1 molar ratio of matrix/analyte. The matrix solution can be adjusted to neutral pH before mixing with the analyte.

[0098] The MALDI ionization surface may be composed of an inert material or else modified to actively capture an analyte. For example, an analyte binding partner may be bound to the surface to selectively absorb a target analyte or the surface may be coated with a thin nitrocellulose film for nonselective binding to the analyte. The surface may also be used as a reaction zone upon which the analyte is chemically modified, e.g., CNBr degradation of protein. See Bai et al, Anal. Chem. 67, 1705-1710 (1995).

[0099] Metals such as gold, copper and stainless steel are typically used to form MALDI ionization surfaces. However, other commercially-available inert materials (e.g., glass, silica, nylon and other synthetic polymers, agarose and other carbohydrate polymers, and plastics) can be used where it is desired to use the surface as a capture region or reaction zone. The use of Nation and nitrocellulose-coated MALDI probes for on-probe purification of PCR-amplified gene sequences is described by Liu et al., Rapid Commun. Mass Spec. 9:735-743 (1995). Tang et al. have reported the attachment of purified oligonucleotides to beads, the tethering of beads to a probe element, and the use of this technique to capture a complimentary DNA sequence for analysis by MALDI-TOF MS (reported by K Tang et al., at the May 1995 TOF-MS workshop, R. J. Cotter (Chairperson); K Tang et al., Nucleic Acids Res. 23, 3126-3131, 1995). Alternatively, the MALDI surface may be electrically- or magnetically activated to capture charged analytes and analytes anchored to magnetic beads respectively.

[0100] Aside from MALDI, Electrospray Ionization Mass Spectrometry (ESI/MS) has been recognized as a significant tool used in the study of proteins, protein complexes and bio-molecules in general. ESI is a method of sample introduction for mass spectrometric analysis whereby ions are formed at atmospheric pressure and then introduced into a mass spectrometer using a special interface. Large organic molecules, of molecular weight over 10,000 Daltons, may be analyzed in a quadrupole mass spectrometer using ESI.

[0101] In ESI, a sample solution containing molecules of interest and a solvent is pumped into an electrospray chamber through a fine needle. An electrical potential of several kilovolts may be applied to the needle for generating a fine spray of charged droplets. The droplets may be sprayed at atmospheric pressure into a chamber containing a heated gas to vaporize the solvent. Alternatively, the needle may extend into an evacuated chamber, and the sprayed droplets are then heated in the evacuated chamber. The fine spray of highly charged droplets releases molecular ions as the droplets vaporize at atmospheric pressure. In either case, ions are focused into a beam, which is accelerated by an electric field, and then analyzed in a mass spectrometer.

[0102] Because electrospray ionization occurs directly from solution at atmospheric pressure, the ions formed in this process tend to be strongly solvated. To carry out meaningful mass measurements, solvent molecules attached to the ions should be efficiently removed, that is, the molecules of interest should be “desolvated.” Desolvation can, for example, be achieved by interacting the droplets and solvated ions with a strong countercurrent flow (6-9 l/m) of a heated gas before the ions enter into the vacuum of the mass analyzer.

[0103] Other well-known ionization methods may also be used. For example, electron ionization (also known as electron bombardment and electron impact), atmospheric pressure chemical ionization (APCI), fast atom Bombardment (FAB), or chemical ionization (CI).

[0104] Immediately following ionization, gas phase ions enter a region of the mass spectrometer known as the mass analyzer. The mass analyzer is used to separate ions within a selected range of mass to charge ratios. This is an important part of the instrument because it plays a large role in the instrument's accuracy and mass range. Ions are typically separated by magnetic fields, electric fields, and/or measurement of the time an ion takes to travel a fixed distance.

[0105] If all ions with the same charge enter a magnetic field with identical kinetic energies a definite velocity will be associated with each mass and the radius will depend on the mass. Thus a magnetic field can be used to separate a monoenergetic ion beam into its various mass components. Magnetic fields will also cause ions to form fragment ions. If there is no kinetic energy of separation of the fragments the two fragments will continue along the direction of motion with unchanged velocity. Generally, some kinetic energy is lost during the fragmentation process creating non-integer mass peak signals which can be easily identified. Thus, the action of the magnetic field on fragmented ions can be used to give information on the individual fragmentation processes taking place in the mass spectrometer.

[0106] Electrostatic fields exert radial forces on ions attracting them towards a common center. The radius of an ion's trajectory will be proportional to the ion's kinetic energy as it travels through the electrostatic field. Thus an electric field can be used to separate ions by selecting for ions that travel within a specific range of radii which is based on the kinetic energy and is also proportion to the mass of each ion.

[0107] Quadrupole mass analyzers have been used in conjunction with electron ionization sources since the 1950s. Quadrupoles are four precisely parallel rods with a direct current (DC) voltage and a superimposed radio-frequency (RF) potential. The field on the quadrupoles determines which ions are allowed to reach the detector. The quadrupoles thus function as a mass filter. As the field is imposed, ions moving into this field region will oscillate depending on their mass-to-charge ratio and, depending on the radio frequency field, only ions of a particular m/z can pass through the filter. The m/z of an ion is therefore determined by correlating the field applied to the quadrupoles with the ion reaching the detector. A mass spectrum can be obtained by scanning the RF field. Only ions of a particular m/z are allowed to pass through.

[0108] Electron ionization coupled with quadrupole mass analyzers can be employed in practicing the instant invention. Quadrupole mass analyzers have found new utility in their capacity to interface with electrospray ionization This interface has three primary advantages. First, quadrupoles are tolerant of relatively poor vacuums (˜5×10−5 torr), which makes it well-suited to electrospray ionization since the ions are produced under atmospheric pressure conditions. Secondly, quadrupoles are now capable of routinely analyzing up to an m/z of 3000, which is useful because electrospray ionization of proteins and other biomolecules commonly produces a charge distribution below m/z 3000. Finally, the relatively low cost of quadrupole mass spectrometers makes them attractive as electrospray analyzers.

[0109] The ion trap mass analyzer was conceived of at the same time as the quadrupole mass analyzer. The physics behind both of these analyzers is very similar. In an ion trap the ions are trapped in a radio frequency quadrupole field. One method of using an ion trap for mass spectrometry is to generate ions externally with ESI or MALDI, using ion optics for sample injection into the trapping volume. The quadrupole ion trap typically consist of a ring electrode and two hyperbolic endcap electrodes. The motion of the ions trapped by the electric field resulting from the application of RF and DC voltages allows ions to be trapped or ejected from the ion trap. In the normal mode the RF is scanned to higher voltages, the trapped ions with the lowest m/z and are ejected through small holes in the endcap to a detector (a mass spectrum is obtained by resonantly exciting the ions and thereby ejecting from the trap and detecting them). As the RF is scanned further, higher m/z ratios become are ejected and detected. It is also possible to isolate one ion species by ejecting all others from the trap. The isolated ions can subsequently be fragmented by collisional activation and the fragments detected. The primary advantages of quadrupole ion traps is that multiple collision-induced dissociation experiments can be performed without having multiple analyzers. Other important advantages include its compact size, and the ability to trap and accumulate ions to increase the signal-to-noise ratio of a measurement.

[0110] Quadrupole ion traps can be used in conjunction with electrospray ionization MS/MS experiments in the instant invention.

[0111] The earliest mass analyzers separated ions with a magnetic field. In magnetic analysis, the ions are accelerated (using an electric field) and are passed into a magnetic field. A charged particle traveling at high speed passing through a magnetic field will experience a force, and travel in a circular motion with a radius depending upon the m/z and speed of the ion. A magnetic analyzer separates ions according to their radii of curvature, and therefore only ions of a given m/z will be able to reach a point detector at any given magnetic field. A primary limitation of typical magnetic analyzers is their relatively low resolution.

[0112] In order to improve resolution, single-sector magnetic instruments have been replaced with double-sector instruments by combining the magnetic mass analyzer with an electrostatic analyzer. The electric sector acts as a kinetic energy filter allowing only ions of a particular kinetic energy to pass through its field, irrespective of their mass-to-charge ratio. Given a radius of curvature, R, and a field, E, applied between two curved plates, the equation R=2V/E allows one to determine that only ions of energy V will be allowed to pass. Thus, the addition of an electric sector allows only ions of uniform kinetic energy to reach the detector, thereby increasing the resolution of the two sector instrument to 100,000. Magnetic double-focusing instrumentation is commonly used with FAB and EI ionization, however they are not widely used for electrospray and MALDI ionization sources primarily because of the much higher cost of these instruments. But in theory, they can be employed to practice the instant invention.

[0113] ESI and MALDI-MS commonly use quadrupole and time-of-flight mass analyzers, respectively. The limited resolution offered by time-of-flight mass analyzers, combined with adduct formation observed with MALDI-MS, results in accuracy on the order of 0.1% to a high of 0.01%, while ESI typically has an accuracy on the order of 0.01%. Both ESI and MALDI are now being coupled to higher resolution mass analyzers such as the ultrahigh resolution (>105) mass analyzer. The result of increasing the resolving power of ESI and MALDI mass spectrometers is an increase in accuracy for biopolymer analysis.

[0114] Fourier-transform ion cyclotron resonance (FTMS) offers two distinct advantages, high resolution and the ability to tandem mass spectrometry experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field. While the ions are orbiting, a radio frequency (RF) signal is used to excite them and as a result of this RF excitation, the ions produce a detectable image current. The time-dependent image current can then be Fourier transformed to obtain the component frequencies of the different ions which correspond to their m/z.

[0115] Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as ±0.001%. The ability to distinguish individual isotopes of a protein of mass 29,000 is demonstrated.

[0116] A time-of-flight (TOF) analyzer is one of the simplest mass analyzing devices and is commonly used with MALDI ionization. Time-of-flight analysis is based on accelerating a set of ions to a detector with the same amount of energy. Because the ions have the same energy, yet a different mass, the ions reach the detector at different times. The smaller ions reach the detector first because of their greater velocity and the larger ions take longer, thus the analyzer is called time-of-flight because the mass is determine from the ions' time of arrival.

[0117] The arrival time of an ion at the detector is dependent upon the mass, charge, and kinetic energy of the ion. Since kinetic energy (KE) is equal to ½ mv2 or velocity v=(2 KE/m)1/2, ions will travel a given distance, d, within a time, t, where t is dependent upon their m/z.

[0118] The magnetic double-focusing mass analyzer has two distinct parts, a magnetic sector and an electrostatic sector. The magnet serves to separate ions according to their mass-to-charge ratio since a moving charge passing through a magnetic field will experience a force, and travel in a circular motion with a radius of curvature depending upon the m/z of the ion. A magnetic analyzer separates ions according to their radii of curvature, and therefore only ions of a given m/z will be able to reach a point detector at any given magnetic field. A primary limitation of typical magnetic analyzers is their relatively low resolution. The electric sector acts as a kinetic energy filter allowing only ions of a particular kinetic energy to pass through its field, irrespective of their mass-to-charge ratio. Given a radius of curvature, R, and a field, E, applied between two curved plates, the equation R=2 V/E allows one to determine that only ions of energy V will be allowed to pass. Thus, the addition of an electric sector allows only ions of uniform kinetic energy to reach the detector, thereby increasing the resolution of the two sector instrument.

[0119] The new ionization techniques are relatively gentle and do not produce a significant amount of fragment ions, this is in contrast to electron ionization (EI) which produces many fragment ions. To generate more information on the molecular ions generated in the ESI and MALDI ionization sources, it has been necessary to apply techniques such as tandem mass spectrometry (MS/MS), to induce fragmentation. Tandem mass spectrometry (abbreviated MSn—where n refers to the number of generations of fragment ions being analyzed) allows one to induce fragmentation and mass analyze the fragment ions. This is accomplished by collisionally generating fragments from a particular ion and then mass analyzing the fragment ions.

[0120] Tandem mass spectrometry or post source decay is used for proteins that cannot be identified by peptide-mass matching or to confirm the identity of proteins that are tentatively identified by an error-tolerant peptide mass search, described above. This method combines two consecutive stages of mass analysis to detect secondary fragment ions that are formed from a particular precursor ion. The first stage serves to isolate a particular ion of a particular peptide (polypeptide) of interest based on its m/z. The second stage is used to analyze the product ions formed by spontaneous or induced fragmentation of the selected ion precursor. Interpretation of the resulting spectrum provides limited sequence information for the peptide of interest. However, it is faster to use the masses of the observed peptide fragment ions to search an appropriate protein sequence database and identify the protein as described in Griffin et al, Rapid Commun. Mass. Spectrom. 1995, 9: 1546. Peptide fragment ions are produced primarily by breakage of the amide bonds that join adjacent amino acids. The fragmentation of peptides in mass spectrometry has been well described (Falick et al., J. Am Soc. Mass Spectrom. 1993, 4, 882-893; Bieniann, K., Biomed. Environ. Mass Spectrom. 1988, 16, 99-111).

[0121] For example, fragmentation can be achieved by inducing ion/molecule collisions by a process known as collision-induced dissociation (CID) or also known as collision-activated dissociation (CAD). CID is accomplished by selecting an ion of interest with a mass filter/analyzer and introducing that ion into a collision cell. A collision gas (typically Ar, although other noble gases can also be used) is introduced into the collision cell, where the selected ion collides with the argon atoms, resulting in fragmentation. The fragments can then be analyzed to obtain a fragment ion spectrum. The abbreviation MSn is applied to processes which analyze beyond the initial fragment ions (MS2) to second (MS3) and third generation fragment ions (MS4). Tandem mass analysis is primarily used to obtain structural information, such as protein or polypeptide sequence, in the instant invention.

[0122] In certain instruments, such as those by JEOL USA, Inc. (Peabody, Mass.), the magnetic and electric sectors in any JEOL magnetic sector mass spectrometer can be scanned together in “linked scans” that provide powerful MS/MS capabilities without requiring additional mass analyzers. Linked scans can be used to obtain product-ion mass spectra, precursor-ion mass spectra, and constant neutral-loss mass spectra These can provide structural information and selectivity even in the presence of chemical interferences. Constant neutral loss spectrum essentially “lifts out” only the interested peaks away from all the background peaks, hence removing the need for class separation and purification. Neutral loss spectrum can be routinely generated by a number of commercial mass spectrometer instruments (such as the one used in the Example section). JEOL mass spectrometers can also perform fast linked scans for GC/MS/MS and LC/MS/MS experiments.

[0123] Once the ion passes through the mass analyzer it is then detected by the ion detector, the final element of the mass spectrometer. The detector allows a mass spectrometer to generate a signal (current) from incident ions, by generating secondary electrons, which are further amplified. Alternatively some detectors operate by inducing a current generated by a moving charge. Among the detectors described, the electron multiplier and scintillation counter are probably the most commonly used and convert the kinetic energy of incident ions into a cascade of secondary electrons. Ion detection can typically employ Faraday Cup, Electron Multiplier, Photomultiplier Conversion Dynode (Scintillation Counting or Daly Detector), High-Energy Dynode Detector (HED), Array Detector, or Charge (or Inductive) Detector.

[0124] The introduction of computers for MS work entirely altered the manner in which mass spectrometry was performed. Once computers were interfaced with mass spectrometers it was possible to rapidly perform and save analyses. The introduction of faster processors and larger storage capacities has helped launch a new era in mass spectrometry. Automation is now possible allowing for thousands of samples to be analyzed in a single day. Te use of computer also helps to develop mass spectra databases which can be used to store experimental results. Software packages not only helped to make the mass spectrometer more user friendly but also greatly expanded the instrument's capabilities.

[0125] The ability to analyze complex mixtures has made MALDI and ESI very useful for the examination of proteolytic digests, an application otherwise known as protein mass mapping. Through the application of sequence specific proteases, protein mass mapping allows for the identification of protein primary structure. Performing mass analysis on the resulting proteolytic fragments thus yields information on fragment masses with accuracy approaching ±5 ppm, or ±0.005 Da for a 1,000 Da peptide. The protease fragmentation pattern is then compared with the patterns predicted for all proteins within a database and matches are statistically evaluated. Since the occurrence of Arg and Lys residues in proteins is statistically high, trypsin cleavage (specific for Arg and Lys) generally produces a large number of fragments which in turn offer a reasonable probability for unambiguously identifying the target protein.

[0126] The primary tools in these protein identification experiments are mass spectrometry, proteases, and computer-facilitated data analysis. As a result of generating intact ions, the molecular weight information on the peptides/proteins are quite unambiguous. Sequence specific enzymes can then provide protein fragments that can be associated with proteins within a database by correlating observed and predicted fragment masses. The success of this strategy, however, relies on the existence of the protein sequence within the database. With the availability of the human genome sequence (which indirectly contain the sequence information of all the proteins in the human body) and genome sequences of other organisms (mouse, rat, Drosophila, C. elegans, bacteria, yeasts, etc.), identification of the proteins can be quickly determined simply by measuring the mass of proteolytic fragments.

[0127] Representative mass spectrometry instruments useful for practicing the instant invention are described in detail in the Examples. A skilled artisan should readily understand that other similar instruments with equivalent function/specification, either commercially available or user modified, are suitable for practicing the instant invention.

[0128] Protease Digestion

[0129] Prior to analysis by mass spectrometry, the protein may be chemically or enzymatically digested. For protein bands from gels, the protein sample in the gel slice may be subjected to in-gel digestion. (see Shevchenko A. et al., Mass Spectrometric Sequencing of Proteins from Silver Stained Polyacrylamide Gels. Analytical Chemistry 1996, 58: 850).

[0130] One aspect of the instant invention is that peptide fragments ending with lysine or arginine residues can be used for sequencing with tandem mass spectrometry. While trypsin is the preferred the protease, many different enzymes can be used to perform the digestion to generate peptide fragments ending with Lys or Arg residues. For instance, in page 886 of a 1979 publication of Enzymes (Dixon, M. et al. ed., 3rd edition, Academic Press, New York and San Francisco, the content of which is incorporated herein by reference), a host of enzymes are listed which all have preferential cleavage sites of either Arg- or Lys- or both, including Trypsin [EC 3.4.21.4], Thrombin [EC 3.4.21.5], Plasmin [EC 3.4.21.7], Kallikrein [EC 3.4.21.8], Acrosin [EC 3.4.21.10], and Coagulation factor Xa [EC 3.4.21.6]. Particularly, Acrosin is the Trypsin-like enzyme of spermatoza, and it is not inhibited by α1-antitrypsin. Plasmin is cited to have higher selectivity than Trypsin, while Thrombin is said to be even more selective. However, this list of enzymes are for illustration purpose only and is not intended to be limiting in any way. Other enzymes known to reliably and predictably perform digestions to generate the polypeptide fragments as described in the instant invention are also within the scope of the invention.

[0131] Sequence and Literature Databases and Database Search

[0132] The raw data of mass spectrometry will be compared to public, private or commercial databases to determine the identity of polypeptides.

[0133] BLAST search can be performed at the NCBI's (National Center for Biotechnology Information) BLAST website. According to the NCBI BLAST website, BLAST® (Basic Local Alignment Search Tool) is a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. The BLAST programs have been designed for speed, with a minimal sacrifice of sensitivity to distant sequence relationships. The scores assigned in a BLAST search have a well-defined statistical interpretation, making real matches easier to distinguish from random background hits. BLAST uses a heuristic algorithm which seeks local as opposed to global alignments and is therefore able to detect relationships among sequences which share only isolated regions of similarity (Altschul et al., 1990, J. Mol. Biol. 215: 403-10). The BLAST website also offer a “BLAST course,” which explains the basics of the BLAST algorithm, for a better understanding of BLAST.

[0134] For protein sequence search, several protein-protein BLAST can be used. Protein BLAST allows one to input protein sequences and compare these against other protein sequences.

[0135] “Standard protein-protein BLAST” takes protein sequences in FASTA format, GenBank Accession numbers or GI numbers and compares them against the NCBI protein databases (see below).

[0136] “PSI-BLAST” (Position Specific Iterated BLAST) uses an iterative search in which sequences found in one round of searching are used to build a score model for the next round of searching. Highly conserved positions receive high scores and weakly conserved positions receive scores near zero. The profile is used to perform a second (etc.) BLAST search and the results of each “iteration” used to refine the profile. This iterative searching strategy results in increased sensitivity.

[0137] “PHI-BLAST” (Pattern Hit Initiated BLAST) combines matching of regular expression pattern with a Position Specific iterative protein search PHI-BLAST can locate other protein sequences which both contain the regular expression pattern and are homologous to a query protein sequence.

[0138] “Search for short, nearly exact sequences” is an option similar to the standard protein-protein BLAST with the parameters set automatically to optimize for searching with short sequences. A short query is more likely to occur by chance in the database. Therefore increasing the Expect value threshold, and also lowering the word size is often necessary before results can be returned. Low Complexity filtering has also been removed since this filters out larger percentage of a short sequence, resulting in little or no query sequence remaining. Also for short protein sequence searches the Matrix is changed to PAM-30 which is better suited to finding short regions of high similarity.

[0139] The databases that can be searched by the BLAST program is user selected, and is subject to frequent updates at NCBI. The most commonly used ones are:

[0140] Nr: All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF;

[0141] Month: All new or revised GenBank CDS translation+PDB+SwissProt+PIR+PRF released in the last 30 days;

[0142] Swissprot: Last major release of the SWISS-PROT protein sequence database (no updates);

[0143] Drosophila genome: Drosophila genome proteins provided by Celera and Berkeley Drosophila Genome Project (BDGP);

[0144]

S. cerevisiae:
Yeast (Saccharomyces cerevisiae) genomic CDS translations;

[0145]

E coli: Escherichia coli
genomic CDS translations;

[0146] Pdb: Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank;

[0147] Alu: Translations of select Alu repeats from REPBASE, suitable for masking Alu repeats from query sequences. It is available by anonymous FTP from the NCBI website. See “Alu alert” by Claverie and Makalowski, Nature vol. 371, page 752 (1994).

[0148] Some of the BLAST databases, like SwissProt, PDB and Kabat are complied outside of NCBI. Other like e coli, dbEST and month, are subsets of the NCBI databases. Other “virtual Databases” can be created using the “Limit by Entrez Query” option.

[0149] The Welcome Trust Sanger Institute offer the Ensembl software system which produces and maintains automatic annotation on eukaryotic genomes. All data and codes can be downloaded without constraints from the Sanger Centre website. The Centre also provides the Ensembl's International Protein Index databases which contain more than 90% of all known human protein sequences and additional prediction of about 10,000 proteins with supporting evidence. All these can be used for database search purposes.

[0150] In addition, many commercial databases are also available for search purposes. For example, Celera has sequenced the whole human genome and offers commercial access to its proprietary annotated sequence database (Discovery™ database).

[0151] Various softwares can be employed to search these databases. The probability search software Mascot (Matrix Science Ltd.). Mascot utilizes the Mowse search algorithm and scores the hits using a probabilistic measure (Perkins et al., 1999, Electrophoresis 20: 3551-3567, the entire contents are incorporated herein by reference). The Mascot score is a function of the database utilized, and the score can be used to assess the null hypothesis that a particular match occurred by chance. Specifically, a Mascot score of 46 implies that the chance of a random hit is less than 5%. However, the total score consists of the individual peptide scores, and occasionally, a high total score can derive from many poor hits. To exclude this possibility, only “high quality” hits—those with a total score >46 with at least a single peptide match with a score of 30 ranking number 1—are considered.

[0152] Other similar softwares can also be used according to manufacturer's suggestion.

[0153] PubMed, available via the NCBI Entrez retrieval system, was developed by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM), located at the National Institutes of Health (NIH). The PubMed database was developed in conjunction with publishers of biomedical literature as a search tool for accessing literature citations and linking to full-text journal articles at web sites of participating publishers.

[0154] Publishers participating in PubMed electronically supply NLM with their citations prior to or at the time of publication. If the publisher has a web site that offers full-text of its journals, PubMed provides links to that site, as well as sites to other biological data, sequence centers, etc. User registration, a subscription fee, or some other type of fee may be required to access the full-text of articles in some journals.

[0155] In addition, PubMed provides a Batch Citation Matcher, which allows publishers (or other outside users) to match their citations to PubMed entries, using bibliographic information such as journal, volume, issue, page number, and year. This permits publishers easily to link from references in their published articles directly to entries in PubMed.

[0156] PubMed provides access to bibliographic information which includes MEDLINE as well as:

[0157] The out-of-scope citations (e.g., articles on plate tectonics or astrophysics) from certain MEDLINE journals, primarily general science and chemistry journals, for which the life sciences articles are indexed for MEDLINE.

[0158] Citations that precede the date that a journal was selected for MEDLINE indexing.

[0159] Some additional life science journals that submit full text to PubMed Central and receive a qualitative review by NLM.

[0160] PubMed also provides access and links to the integrated molecular biology databases included in NCBI's Entrez retrieval system. These databases contain DNA and protein sequences, 3-D protein structure data, population study data sets, and assemblies of complete genomes in an integrated system.

[0161] MEDLINE is the NLM's premier bibliographic database covering the fields of medicine, nursing, dentistry, veterinary medicine, the health care system, and the pre-clinical sciences. MEDLINE contains bibliographic citations and author abstracts from more than 4,300 biomedical journals published in the United States and 70 other countries. The file contains over 11 million citations dating back to the mid-1960's. Coverage is worldwide, but most records are from English-language sources or have English abstracts.

[0162] PubMed's in-process records provide basic citation information and abstracts before the citations are indexed with NLM's MeSH Terms and added to MEDLINE. New in process records are added to PubMed daily and display with the tag [PubMed—in process]. After MeSH terms, publication types, GenBank accession numbers, and other indexing data are added, the completed MEDLINE citations are added weekly to PubMed.

[0163] Citations received electronically from publishers appear in PubMed with the tag [PubMed—as supplied by publisher]. These citations are added to PubMed Tuesday through Saturday. Most of these progress to In Process, and later to MEDLINE status. Not all citations will be indexed for MEDLINE and are tagged, [PubMed—as supplied by publisher].

[0164] The Batch Citation Matcher allows users to match their own list of citations to PubMed entries, using bibliographic information such as journal, volume, issue, page number, and year. The Citation Matcher reports the corresponding PMID. This number can then be used to easily to link to PubMed. This service is frequently used by publishers or other database providers who wish to link from bibliographic references on their web sites directly to entries in PubMed.

[0165] Separation of Polypeptide Complexes

[0166] Polypeptide separation schemes can achieved based on differences in the molecular properties such as size, charge and solubility. Protocols based on these parameters include SDS-PAGE (SDS-PolyAcrylamide Gel Electrophoresis), size exclusion chromatography, ion exchange chromatography, differential precipitation and the like. SDS-PAGE is well-known in the art of biology, and will not be described here in detail. See Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989).

[0167] Size exclusion chromatography, otherwise known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration is a function of the hydrodynamic volume of the particles. Accordingly, under ideal conditions the larger molecules are excluded from the interior of the particles while the smaller molecules are accessible to this volume and the order of elution can be predicted by the size of the polypeptide because a linear relationship exists between elution volume and the log of the molecular weight. Size exclusion chromatographic supports based on cross-linked dextrans e.g. SEPHADEX.RTM., spherical agarose beads e.g. SEPHAROSE.RTM. (both commercially available from Pharmacia AB. Uppsala, Sweden), based on cross-linked polyacrylamides e.g. BIO-GEL.RTM. (commercially available from BioRad Laboratories, Richmond, Calif.) or based on ethylene glycol-methacrylate copolymer e.g. TOYOPEARL HW65S (commercially available from ToyoSoda Co., Tokyo, Japan) are useful in the practice of this invention.

[0168] Precipitation methods are predicated on the fact that in crude mixtures of polypeptides the solubilities of individual polypeptides are likely to vary widely. Although the solubility of a polypeptide in an aqueous medium depends on a variety of factors, for purposes of this discussion it can be said generally that a polypeptide will be soluble if its interaction with the solvent is stronger than its interaction with polypeptide molecules of the same or similar kind. Without wishing to be bound by any particular mechanistic theory describing precipitation phenomena, it is nonetheless believed that the interaction between a polypeptide and water molecules occur by hydrogen bonding with several types of charged groups, and electrostatically as dipoles with uncharged groups, and that precipitants such as salts of monovalent cations (e.g., ammonium sulfate) compete with polypeptides for water molecules, thus at high salt concentrations, the polypeptides become “dehydrated” reducing their interaction with the aqueous environment and increasing the aggregation with like or similar polypeptides resulting in precipitation from the medium.

[0169] Ion exchange chromatography involves the interaction of charged functional groups in the sample with ionic functional groups of opposite charge on an adsorbent surface. Two general types of interaction are known. Anionic exchange chromatography mediated by negatively charged amino acid side chains (e.g. aspartic acid and glutamic acid) interacting with positively charged surfaces and cationic exchange chromatography mediated by positively charged amino acid residues (e.g. lysine and arginine) interacting with negatively charged surfaces.

[0170] More recently affinity chromatography and hydrophobic interaction chromatography techniques have been developed to supplement the more traditional size exclusion and ion exchange chromatographic protocols. Affinity chromatography relies on the interaction of the polypeptide with an immobilized ligand. The ligand can be specific for the particular polypeptide of interest in which case the ligand is a substrate, substrate analog, inhibitor or antibody. Alternatively, the ligand may be able to react with a number of polypeptides. Such general ligands as adenosine monophosphate, adenosine diphosphate, nicotine adenine dinucleotide or certain dyes may be employed to recover a particular class of polypeptides. One of the least biospecific of the affinity chromatographic approaches is immobilized metal affinity chromatography (IMAC), also referred to as metal chelate chromatography. IMAC introduced by Porath et al.(Nature 258:598-99(1975) involves chelating a metal to a solid support and then forming a complex with electron donor amino acid residues on the surface of a polypeptide to be separated.

[0171] Hydrophobic interaction chromatography was first developed following the observation that polypeptides could be retained on affinity gels which comprised hydrocarbon spacer arms but lacked the affinity ligand. Although in this field the term hydrophobic chromatography is sometimes used, the term hydrophobic interaction chromatography (HIC) is preferred because it is the interaction between the solute and the gel that is hydrophobic not the chromatographic procedure. Hydrophobic interactions are strongest at high ionic strength, therefore, this form of separation is conveniently performed following salt precipitations or ion exchange procedures. Elution from HIC supports can be effected by alterations in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene glycol. A description of the general principles of hydrophobic interaction chromatography can be found in U.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098. The application of HIC to the purification of specific polypeptides is exemplified by reference to the following disclosures: human growth hormone (U.S. Pat. No. 4,332,717), toxin conjugates (U.S. Pat. No. 4,771,128), antihemolytic factor (U.S. Pat No. 4,743,680), tumor necrosis factor (U.S. Pat. No. 4,894,439), interleukin-2 (U.S. Pat. No. 4,908,434), human lymphotoxin (U.S. Pat. No. 4,920,196) and lysozyme species (Fausnaugh, J. L. and F. E. Regnier, J. Chromatog. 359:131-146 (1986)).

[0172] The principles of IMAC are generally appreciated. It is believed that adsorption is predicated on the formation of a metal coordination complex between a metal ion, immobilized by chelation on the adsorbent matrix, and accessible electron donor amino acids on the surface of the polypeptide to be bound. The metal-ion microenvironment including, but not limited to, the matrix, the spacer arm, if any, the chelating ligand, the metal ion, the properties of the surrounding liquid medium and the dissolved solute species can be manipulated by the skilled artisan to affect the desired fractionation.

[0173] Not wishing to be bound by any particular theory as to mechanism, it is further believed that the more important amino acid residues in terms of binding are histidine, tryptophan and probably cysteine. Since one or more of these residues are generally found in polypeptides, one might expect all polypeptides to bind to IMAC columns. However, the residues not only need to be present but also accessible (e.g., oriented on the surface of the polypeptide) for effective binding to occur. Other residues, for example poly-histidine tails added to the amino terminus or carboxyl terminus of polypeptides, can be engineered into the recombinant expression systems by following the protocols described in U.S. Pat No. 4,569,794.

[0174] The nature of the metal and the way it is coordinated on the column can also influence the strength and selectivity of the binding reaction. Matrices of silica gel, agarose and synthetic organic molecules such as polyvinyl-methacrylate co-polymers can be employed. The matrices preferably contain substituents to promote chelation. Substituents such as iminodiacetic acid (IDA) or its tris (carboxymethyl) ethylene diamine (TED) can be used. IDA is preferred. A particularly useful IMAC material is a polyvinyl methacrylate co-polymer substituted Keith IDA available commercially, e.g., as TOYOPEARL AF-CHELATE 650M (ToyoSoda Co.; Tokyo. The metals are preferably divalent members of the first transition series through to zinc, although Co++, Ni++, Cd++ and Fe+++ can be used. An important selection parameter is, of course, the affinity of the polypeptide to be purified for the metal. Of the four coordination positions around these metal ions, at least one is occupied by a water molecule which is readily replaced by a stronger electron donor such as a histidine residue at slightly alkaline pH.

[0175] In practice the IMAC column is “charged” with metal by pulsing with a concentrated metal salt solution followed by water or buffer. The column often acquires the color of the metal ion (except for zinc). Often the amount of metal is chosen so that approximately half of the column is charged. This allows for slow leakage of the metal ion into the non-charged area without appearing in the eluate. A pre-wash with intended elution buffers is usually carried out. Sample buffers may contain salt up to 1M or greater to minimize nonspecific ion-exchange effects. Adsorption of polypeptides is maximal at higher pHs. Elution is normally either by lowering of pH to protonate the donor groups on the adsorbed polypeptide, or by the use of stronger complexing agent such as imidazole, or glycine buffers at pH 9. In these latter cases the metal may also be displaced from the column. Linear gradient elution procedures can also be beneficially employed.

[0176] As mentioned above, IMAC is particularly useful when used in combination with other polypeptide fractionation techniques. That is to say it is preferred to apply IMAC to material that has been partially fractionated by other protein fractionation procedures. A particularly useful combination chromatographic protocol is disclosed in U.S. Pat. No. 5,252,216 granted Oct. 12, 1993, the contents of which are incorporated herein by reference. It has been found to be useful, for example, to subject a sample of conditioned cell culture medium to partial purification prior to the application of IMAC. By the term “conditioned cell culture medium” is meant a cell culture medium which has supported cell growth and/or cell maintenance and contains secreted product. A concentrated sample of such medium is subjected to one or more polypeptide purification steps prior to the application of a IMAC step. The sample may be subjected to ion exchange chromatography as a first step. As mentioned above various anionic or cationic substituents may be attached to matrices in order to form anionic or cationic supports for chromatography. Anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K SEPHADEX.RTM.-based and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-dextran supports under the tradename SEPHADEX.RTM. and DEAE-, Q-, CM-and S-agarose supports under the tradename SEPHAROSE.RTM. are all available from Pharmacia AB. Further both DEAE and CM derivitized ethylene glycol-methacrylate copolymer such as TOYOPEARL DEAE-650S and TOYOPEARL CM-650S are available from Toso Haas Co., Philadelphia, Pa. Because elution from ionic supports sometimes involves addition of salt and IMAC may be enhanced under increased salt concentrations. The introduction of a IMAC step following an ionic exchange chromatographic step or other salt mediated purification step may be employed. Additional purification protocols may be added including but not necessarily limited to HIC, further ionic exchange chromatography, size exclusion chromatography, viral inactivation, concentration and freeze drying.

[0177] Hydrophobic molecules in an aqueous solvent will self-associate. This association is due to hydrophobic interactions. It is now appreciated that macromolecules such as polypeptides have on their surface extensive hydrophobic patches in addition to the expected hydrophilic groups. HIC is predicated, in part, on the interaction of these patches with hydrophobic ligands attached to chromatographic supports. A hydrophobic ligand coupled to a matrix is variously referred to herein as an HIC support, HIC gel or HIC column. It is further appreciated that the strength of the interaction between the polypeptide and the HIC support is not only a function of the proportion of non-polar to polar surfaces on the polypeptide but by the distribution of the non-polar surfaces as well.

[0178] A number of matrices may be employed in the preparation of HIC columns, the most extensively used is agarose. Silica and organic polymer resins may be used. Useful hydrophobic ligands include but are not limited to alkyl groups having from about 2 to about 10 carbon atoms, such as a butyl, propyl, or octyl; or aryl groups such as phenyl. Conventional HIC products for gels and columns may be obtained commercially from suppliers such as Pharmacia LKB AB, Uppsala, Sweden under the product names butyl-SEPHAROSE.RTM., phenyl-SEPHAROSE.RTM. CL-4B, octyl-SEPHAROSE.RTM. FF and phenyl-SEPHAROSE.RTM. FF; Tosoh Corporation, Tokyo, Japan under the product names TOYOPEARL Butyl 650, Ether-650, or Phenyl-650 (FRACTOGEL TSK Butyl-650) or TSK-GEL phenyl-5PW; Miles-Yeda, Rehovot, Israel under the product name ALKYL-AGAROSE, wherein the alkyl group contains from 2-10 carbon atoms, and J. T. Baker, Phillipsburg, N.J. under the product name BAKERBOND WP-HI-propyl.

[0179] Ligand density is an important parameter in that it influences not only the strength of the interaction but the capacity of the column as well. The ligand density of the commercially available phenyl or octyl phenyl gels is on the order of 40 μM/ml gel bed. Gel capacity is a function of the particular polypeptide in question as well pH, temperature and salt concentration but generally can be expected to fall in the range of 3-20 mg/ml of gel.

[0180] The choice of a particular gel can be determined by the skilled artisan. In general the strength of the interaction of the polypeptide and the HIC ligand increases with the chain length of the of the alkyl ligands but ligands having from about 4 to about 8 carbon atoms are suitable for most separations. A phenyl group has about the same hydrophobicity as a pentyl group, although the selectivity can be quite different owing to the possibility of pi-pi interaction with aromatic groups on the polypeptide.

[0181] Adsorption of the polypeptides to a HIC column is favored by high salt concentrations, but the actual concentrations can vary over a wide range depending on the nature of the polypeptide and the particular HIC ligand chosen. Various ions can be arranged in a so-called soluphobic series depending on whether they promote hydrophobic interactions (salting-out effects) or disrupt the structure of water (chaotropic effect) and lead to the weakening of the hydrophobic interaction. Cations are ranked in terms of increasing salting out effect as Ba++<Ca++<Mg++<Li+<Cs+<Na+<K+<Rb+<NH4+. While anions may be ranked in terms of increasing chaotropic effect as PO4−−−<SO4−−<CH3COO−<Cl−<Br−<NO3−<CIO4−<I−<SCN−.

[0182] Accordingly, salts may be formulated that influence the strength of the interaction as given by the following relationship:

Na2SO4>NaCl>(NH4)2SO4>NH4Cl>NaBr>NaSCN

[0183] In general, salt concentrations of between about 0.75 and about 2M ammonium sulfate or between about 1 and 4M NaCl are useful.

[0184] The influence of temperature on HIC separations is not simple, although generally a decrease in temperature decreases the interaction However, any benefit that would accrue by increasing the temperature must also be weighed against adverse effects such an increase may have on the activity of the polypeptide.

[0185] Elution, whether stepwise or in the form of a gradient, can be accomplished in a variety of ways: (a) by changing the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents. By decreasing salt concentration adsorbed polypeptides are eluted in order of increasing hydrophobicity. Changes in polarity may be affected by additions of solvents such as ethylene glycol or (iso)propanol thereby decreasing the strength of the hydrophobic interactions. Detergents function as displacers of polypeptides and have been used primarily in connection with the purification of membrane polypeptides.

[0186] When the eluate resulting from HIC is subjected to further ion exchange chromatography, both anionic and cationic procedures may be employed.

[0187] As mentioned above, gel filtration chromatography affects separation based on the size of molecules. It is in effect a form of molecular sieving. It is desirable that no interaction between the matrix and solute occur, therefore, totally inert matrix materials are preferred. It is also desirable that the matrix be rigid and highly porous. For large scale processes rigidity is most important as that parameter establishes the overall flow rate. Traditional materials such as crosslinked dextran or polyacrylamide matrices, commercially available as, e.g., SEPHADEX.RTM. and BIOGEL.RTM., respectively, were sufficiently inert and available in a range of pore sizes, however these gels were relatively soft and not particularly well suited for large scale purification. More recently, gels of increased rigidity have been developed (e.g. SEPHACRYL.RTM., ULTROGEL.RTM., FRACTOGEL.RTM. and SUPEROSE.RTM.). All of these materials are available in particle sizes which are smaller than those available in traditional supports so that resolution is retained even at higher flow rates. Ethylene glycol-methacrylate copolymer matrices, e.g., such as the TOYOPEARL HW series matrices (Toso Haas) are preferred.

[0188] Phosphoproteins can be isolated using IMAC as described above. However, they can also be isolated by other means. Specifically, phosphoproteins with phosphorylated tyrosine residues can be isolated with phospho-tyrosine specific antibodies. Likewise, phospho-serine/threonine specific antibodies can be used to isolate phosphoproteins with phosphorylated serine/threonine residues. Many of these antibodies are available as affinity purified forms, either as monoclonal antibodies or antisera or mouse ascites fluid. For example, phospho-Tyrosine monoclonal antibody (P-Tyr-102) is a high-affinity IgG1 phospho-tyrosine antibody clone that is produced and characterized by Cell Signaling Technology (Beverly, Mass.). As determined by ELISA, P-Tyr-102 (Cat. No. 9416) binds to a larger number of phospho-tyrosine containing peptides in a manner largely independent of the surrounding amino acid sequences, and also interacts with a broader range of phospho-tyrosine containing polypeptides as indicated by 2D-gel Western analysis. P-Tyr-102 is highly specific for phospho-Tyr in peptides/proteins, shows no cross-reactivity with the corresponding nonphosphorylated peptides and does not react with peptides containing phospho-Ser or phospho-Thr instead of phospho-Tyr. It is expected that P-Tyr-102 will react with peptides/proteins containing phospho-Tyr from all species.

[0189] Phospho-threonine antibodies are also available. For example, Cell Signaling Technology also offer an affinity-purified rabbit polyclonal phospho-threonine antibody (P-Thr-Polyclonal, Cat. No. 9381) which binds threonine-phosphorylated sites in a manner largely independent of the surrounding amino acid sequence. It recognizes a wide range of threonine-phosphorylated peptides in ELISA and a large number of threonine-phosphorylated polypeptides in 2D analysis. It is specific for peptides/proteins containing phospho-Thr and shows no cross-reactivity with corresponding nonphosphorylated sequences. Phospho-Threonine Antibody (P-Thr-Polyclonal) does not cross-react with sequences containing either phospho-Tyrosine or phospho-Serine. It is expected that this antibody will react with threonine-phosphorylated peptides/proteins regardless of species of origin. Upstate Biotechnology (Lake Placid, N.Y.) also provides an anti-phospho-serine/threonine antibody with broad immunoreactivity for polypeptides containing phosphorylated serine and phosphorylated threonine residues.

[0190] Many other similar products are also available on the market. These antibodies can be readily coupled to supporting matrix materials to generate affinity columns according to standard molecular biology protocols (for details and general means of antibody production, see Using Antibodies: A Laboratory Manual: Portable Protocol NO. I, Harlow and Lane, Cold Spring Harbor Laboratory Press: 1998; also see Antibodies : A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor Laboratory Press: 1988).

[0191] A similar approach can be applied towards the isolation of any specific polypeptide, against which specific antibodies are available.

[0192] Isolation of membrane-associated polypeptides can be carried out using appropriate methods as described above (for example, hydrophobic interaction chromatography). Alternatively, it can be performed with other standard molecular biology protocols. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987).

[0193] For example, cells can be lysed in appropriate buffers and the membrane portions can be isolated by centrifugation. Depending on particular cases, cells preferably can be lysed in hypotonic buffer by homogenization. Cell debris and nuclei can then be removed by low speed centrifugation, followed by high speed centrifugation (such as under centrifugation conditions of 100,000×g or more) to pellet membrane portions. Membrane polypeptides can then be extracted by organic solvents such as chloroform and methanol.

[0194] Alternatively, membrane polypeptides can be isolated by extraction of membrane portions with extraction buffer containing detergents. Depending on specific occasions, the detergent used can be SDS or other ionic or non-ionic detergents. Different choices of detergent or extraction buffer in general may facilitate global non-biased extraction of membrane polypeptides or isolation of specific membrane polypeptides of interest. The reduced complexity of polypeptide mixtures resulting from the use of specific extraction protocols may be beneficial for the following digestion, separation, and analysis procedures.

[0195] A most preferred method of isolating hydrophobic membrane proteins is strong cation exchange (SCX) chromatography. Strong cation exchange (SCX) chromatography is particularly suited for isolating/purifying hydrophobic proteins, such as membrane proteins. Many SCX chromatographic columns are commercially available. For illustration purpose only, details regarding one type of SCX column, the PolySulfoethyl Aspartamide Strong Cation Exchange Columns manufactured by The Nest Group, Inc. (45 Valley Road, Southborough, Mass.), are described below. It is to be understood that the recommendations below are by no means limiting in any respect. Many other commercial SCX columns are also available, and should be used according to the recommendation of respective manufacturers.

[0196] According to the manufacturer, aspartamide cation exchange chemistries are some of the best materials available for the HPLC separation of peptides. These are wide-pore (300 Å) silica packings with a bonded coating of hydrophilic, sulfoethyl anionic polymer. With the PolySULFOETHYL Aspartamide SCX column, mobile phase modifiers can be used to help improve peptide solubility or to mediate the interaction between peptide and stationary phase. By varying the pH, ionic strength or organic solvent concentration in the mobile phase, chromatographic selectivity can be significantly enhanced. For more strongly hydrophobic peptides, a non-ionic surfactant (at a concentration below its CMC) and/or acetonitrile or n-propanol as mobile phase modifiers, can substantially improve resolution and recovery over conventional reverse phase methods. Additional selectivity can be obtained by simply changing the slope of the KCl or (NH4)2SO4 gradient.

[0197] Using this column at pH 3 is better for retention of neutral to slightly acidic peptides. Use of a higher pH may be considered for basic hydrophobic peptides. The addition of MeCN or propanol to the A&B solvents (see below) changes the mechanism of separation and results in a separation based not only on positive charge, but also on hydrophobicity.

[0198] These columns are quite useful for neuropeptides, growth factors, CNBr peptide fragments, and synthetic peptides as a complement to RPC (Reverse Phase Chromatography), or to remove organic reagents from peptide samples which would cause smearing on a RPC column.

[0199] The operating conditions for these applications for an analytical column are:

[0200] Buffer A: 5 mM K-PO4+25% MeCN;

[0201] Buffer B: 5 mM K-PO4+25% MeCN+300-500 mM KCl;

[0202] Linear gradient, 30 min at 1 ml/min.

[0203] The peptides are retained on the column by the positive charge of at least the terminus amino and elute by total charge, charge distribution and hydrophobicity. If the peptide does not stick to the column, prepare the peptide in a small amount of buffer, or decrease the concentration of organic in the A&B solvents to 5 or 10%. Organic solvent concentration is empirically determined and n-propanol can be substituted for MeCN for more hydrophobic species.

[0204] Since the total binding capacity of these columns is on the order of 100 mg/gm of packing (for nonresolved materials) there will be a considerable Donan effect present. It will be necessary to have the sample in 5-15 mM of salt or buffer to prevent exclusion from the column. Additionally, the gradient at the outlet of the column will be much more concave than that observed on the chart paper. It is recommended that an upper load limit of 1 milligram for an analytical column. For a guard column used as a methods development column, a load limit of one-tenth of a milligram is recommended.

[0205] Flow rates of 0.7 to 1.0 ml/min with a 30 minutes gradient should be used for the analytical column. If using the 4.6×20 mm guard column as a methods development column, gradient times should be shortened to 8-10 min at the same flow rate since the void volume is only 0.3 ml. The semiprep columns, 9.4 mm ID, require flow rates and equilibration volumes 4× that of the analytical columns.

[0206] Typically, for the first run, equilibrate the analytical column in the high salt (or final pH) solution (at least 25 ml, or for a guard column used as a methods development column use 8 ml, or on the semiprep column use 100 ml), and inject the sample under these isocratic conditions to observe the elution profile. The protein should elute at the void volume. Then equilibrate the column in low salt (or low pH if doing a pH gradient) conditions and run the gradient to the final conditions. Comparison of the chromatograms will assure that the proteins will elute in a predictable fashion. To decrease elution times increase the salt concentration (in a convex or step manner), increase the pH, or shorten the equilibration times between gradient runs. Exposure to a pH above 7 should be avoided since this will affect the silica support and will shorten column life, as will temperatures above 45° C. For buffer gradients, phosphate or bis-tris are good buffers to use since they allow monitoring in the low UV range. For salt gradients, acetate salts are frequently used. However, it may be necessary to use sulfate or chloride if the buffering capacity of acetate is undesirable or if the absorbance is to be monitored below 235 nm. When chloride has been used for salt gradient elution, flush the column with at least 30 ml of deionized water at the end of the day to prevent corrosion. If a denaturant such as 4M urea is used in the mobile phase to increase the accessibility of the ionizable groups, be sure to have a silica saturator column in line in front of the injector, to minimize attack of the silica on the ion exchange column.

[0207] New columns should be condition before use, preferably according to the following protocol. Specifically, columns are filled with methanol when shipped so the (analytical) column should be flushed with at least 40 ml water before elution with salt solution to prevent precipitation The hydrophilic coating imbibes a layer of water. The resultant swelling of the coating leads to a slight and irreversible increase in the column back pressure. Some additional swelling occurs with extended use of the column. Since the swelling increases the surface area of the coating, the capacity of the column for proteins increases as well. Thus, retention times may increase by up to 10%. This process should be hastened by eluting the column with a strong buffer for at least one hour prior to its initial use. A convenient solution to use is 0.2 M monosodium phosphate +0.3 M sodium acetate.

[0208] The conditioning process is reversed by exposing the column to pure organic solvents. Accordingly, to minimize the time to start the column after a 1-2 day storage, the column should be flushed with at least 40 ml of deionized water (not methanol), and the ends should be plugged. For extended storage it is recommended that a 100% methanol storage be used to prevent bacterial growth and contamination. Exercise care when using organic solvents to prevent precipitation of salts.

[0209] It is recommended that a new column be conditioned with two injections of an inexpensive protein (e.g. BSA) before it is used to analyze very dilute or expensive samples since new HPLC columns sometimes absorb small quantities of proteins in a nonspecific manner. The sintered metal frits have been implicated in this process. Fortunately these sites are quickly saturated. Mobile phases should be filtered before use, as should samples. Failure to do so may cause the inlet frit to plug. A guard column, P410-2SEA, will prevent damage to the analytical or preparative columns. Use of 0.1% TFA or high concentrations of formic acid in the mobile phase is not recommended.

[0210] For use in normal phase and HILIC polarity, the following should be taken into consideration. By adding even more organic solvent to the mobile phase, these columns offer enough flexibility so that they may be used in a normal or Hydrophilic Interaction (HILIC) mode. Here, more polar peptides having little or no retention under conventional reverse-phase or even ion-exchange conditions are retained, and very hydrophobic peptides may have enhanced solubility and thus chromatograph better. There are two approaches to this mode: 1) using isocratic HILIC conditions or 2) using a sodium perchlorate gradient. The key to achieving HILIC conditions is to use greater than 70% organic solvent with the SCX column. Care should be taken to assure solubility of salts under these conditions.

[0211] Automation and High Throughput Screening

[0212] The methods of the present invention may be conducted in a high throughput fashion and/or by automation. One non-limiting example of high throughput is repeating a method, or variations of a method, a substantial number of times more quickly than would be possible using standard laboratory techniques. In many instances, the method is used with different samples. By a high throughput method, a single or several individuals may process about 5, 10, 25, 50, 75, 100, 250, 500, 750, 1000, 5000, or 10,000 times the number of samples than the same number of individuals would be able to process in the same time period (one, three, seven, 30, 60, 90 days).

[0213] Automation has been used to achieve high throughput. In regard to automation of the present subject methods, a variety of instrumentation may be used. In general, automation, as used in reference to the subject method, involves having instrumentation complete one or more of the operative steps that must be repeated a multitude of times in performing the method with different samples. Examples of automation include, without limitation, having instrumentation complete coupling of anti-tag antibodies to a solid support, adding the extract to an assay environment or other vessel, washings, loading of samples for separation followed by mass spectrometry of eluted polypeptides, and data collection/analysis, etc.

[0214] There is a range of automation possible for the present invention For example, the subject methods may be wholly automated or only partially automated. If wholly automated, the method may be completed by the instrumentation without any human intervention after initiating it, other than refilling reagent bottles or monitoring or programming the instrumentation as necessary. In contrast, partial automation of the subject method involves some robotic assistance with the physical steps of the method, such as mixing, washing and the like, but still requires some human intervention other than just refilling reagent bottles or monitoring or programming the instrumentation.

[0215] For example, in a preferred embodiment, the methods of the instant invention may be performed in a modular fashion. Specifically, it may include: (a) a module for retrieving recombinant clones encoding bait proteins; (b) an automated immunoprecipitation module for purification of complexes comprising bait and prey proteins; (c) an analysis module for further purifying the proteins from (b) or preparing fragments of such proteins that are suitable for mass spectrometry; (d) a mass spectrometer module for automated analysis of fragments from (c); (d) a computer module comprising an integration software for communication among the modules of the system and integrating operations; and (e) a module for performing an automated method of the invention.

[0216] Several computer implemented methods for managing HTS-process information are known. Most automated lab systems have software that takes care of scheduling samples through the system. The technician sets up the scientific method to be executed. These methods denote the exact steps that are to be performed on a single sample. A technician then executes a scheduling algorithm on a particular number of samples which determines the sample step interleaving. These scheduler must balance the load, prevent deadlocks and enforce resource use and availability.

[0217] Automated lab systems today are known as Laboratory Information Management Systems (LIMS). LIMS typically involve the integration of automated robots into a central computing system allowing for control of the processes of each work-unit involved. An example of such a LIMS is described in U.S. Pat. No. 5,985,214 (incorporated herein by reference) wherein a system and a method for rapidly identifying chemicals in liquid samples is described. The system focuses on the rapid processing of addressable sample wells or the routing of these addressable wells.

[0218] LIMS typically include sample automation and data automation. Sample automation primarily involves control of robotics processes, routing of samples and sample tracking. Data automation typically involves generation of data accumulated from a wide variety of sources. WO 99/05591 (incorporated herein by reference) describes a system and method for organizing information relating to polymer probe array chips whereby a database model is provided which organizes information relating to sample preparation, chip layout, application of samples to chips, scanning of chips, expression analysis of chip results, etc. This system models the specific high throughput entities as if the testing would be performed manually. WO 02/065334 A1 (incorporated herein by reference) provides a computer-implemented method for managing information relating to a high throughput screening (HTS) process and to apparatuses or robot means controlled by said method. A database model is provided which organizes information relating to analytes, biological targets, HTS supports, HTS conditions, interaction results, robotics steering and control, etc. WO 02/49761 A2 (incorporated herein by reference) also provides an automated laboratory system and method allow high-throughput and fully automated processing of materials, such as liquids including genetic materials. It includes a variety of aspects that may be combined into a single system. For example, processing may be performed by a plurality of robotic-equipped modular stations, where each modular station has its own unique environment in which processes are performed. Transport devices, such as conveyor belts, may move objects between modular stations, saving movement for robots in the modular stations. Gels used for gel electrophoresis may be extruded, thus decreasing the time needed to form such gels. Robotically-operated well forming tools allow wells to be formed in gels in a registered and accurate way.

[0219] WO 02/068157 A2 provides grasping mechanisms, gripper apparatus/systems, and related methods, which is useful for accurate positioning of an object (such as a microtiter plate) for automated processing. Grasping mechanisms that include stops, support surfaces, and height adjusting surfaces to determine three translational axis positions of a grasped object are provided. In addition, grasping mechanisms that are resiliently coupled to other gripper apparatus components are also provided.

[0220] Steps related to the invention, as well as alternative means of accomplishing the same or similar goals are illustrated herein. Although yeast was used in the example that follows, it should also be noted that such technique is not limited to yeast. With minor modification, very similar procedures as described below can be used for similar assays in higher eukaryotes, including mammalian cells, such as human cells.

[0221] The following non-limiting example is illustrative of the present invention.

EXAMPLE

[0222] The following materials and methods were used in the studies described in the Example:

[0223] Materials and Methods

[0224] The base vector used for the example shown below, MT2250, was constructed as follows. FLAG-tagged yeast open reading frames (ORFs) were cloned using the Gateway™ recombination-based cloning system (Invitrogen). A galactose-inducible, C-terminal FLAG tag Gateway™ destination vector, called pGAL1-CFLAG, was constructed by inserting annealed FLAG-1/2 oligonucleotides (FLAG-1: 5′-GATCCCCCGGGATGGATTACAAGGATGACGA-CGATAAGTAACTGCA-3′ (SEQ ID NO: 1), FLAG-2: 5′-GTTATCCGCCCGG-GCTCTTATCGTCGTCATCCTTGTAATCCATCCCGGGG-3′ (SEQ ID NO: 2); FLAG=DYKDDDDL (SEQ ID NO: 3), Sigma-Aldrich) into a <GAL1 LEU2 CEN> base vector (MT2250) cut with BamHI and PstI, followed by insertion of conversion cassette B into the SmaI site. A doxycyclin-inducible C-terminal FLAG tag Gateway™ destination vector, called ptet-CFLAG, was constructed by inserting the conversion cassette B-FLAG tag region from pGAL1-CFLAG, removed as a SpeI-ClaI fragment, into pCM251 between the BamHI site and ClaI site2. Both donor vectors were propagated in the E. coli DB3.1 strain to prevent lethality of the ccdB gene in the Gateway™ conversion cassette. Yeast ORFs were amplified by PCR using a 5′ primer that included the attB1 recombinational site (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTA-3′, SEQ ID NO: 4), followed by the start codon and 18-24 bp of gene-specific sequence and a 3′ primer that included the attB2 recombinational site (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTC-3′, SEQ ID NO: 5) followed by 18-24 bp of gene-specific sequence immediately upstream of the stop codon. PCR amplification was performed with Platinum Taq Hi Fidelity DNA polymerase protocol using 100 ng of S288C yeast genomic DNA. PCR products were purified using a Millipore Multiscreen-PCR system and inserted into pGAL1-CFLAG using recombinational cloning as recommended (Invitrogen).

[0225] Proteins cloned using vectors such as this, and subsequently expressed in suitable hosts, are used as bait proteins.

[0226] Yeast Culture

[0227] The yeast strains used in this study were YP1 and YP2. YP1 was strain BY4472 pep4ΔkanR from the deletion consortium (Winzeler, 1999). Strain YP2 was strain YP1 deleted for TRP1 using the plasmid, pTH4 which replaces the TRP1 gene with the HIS3 gene so that the resulting strain in trp−, HIS+ (Cross, 1997). General yeast biology techniques are common knowledge and will not be recited. XY medium contains 2% bactopeptone, 1% yeast extract, 0.01% adenine, 0.02% tryptophan.

[0228] Capture of Protein Complexes

[0229] Strain BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pep4Δ:KANR from the international yeast deletion consortium, or a variant strain YP2 (BY4742 pep4Δ::KANR trp1Δ:HIS3) were used for protein expression. Yeast biology techniques were essentially used as described. XY medium contains 2% bactopeptone, 1% yeast extract, 0.01% adenine, 0.02% tryptophan. To overcome difficulties in expression, such as for poorly expressed genes or developmentally regulated genes, all baits were expressed from either the inducible GAL1 or tet promoters for short induction periods. Although this approach is subject to caveat of over-expression, we minimized such effects by using short induction periods, typically 1-2 hours. The tet promoter was also used for some experiments. Other inducible systems are also generally available for this purpose. To maximize recovery of delicate protein complexes, we utilized concentrated cell extracts, from which the FLAG epitope could be captured with 50-100% efficiency (data not shown). Yeast culture volumes of 500 mL or less were used to prepare cell extracts for capture on anti-FLAG resin (Sigma-Aldrich), according to either protocol A or protocol B, as follows.

[0230] Protocols A and B were done over two physical locations.

[0231] Protocol A: BY4742 bearing pGAL1-CFLAG expressing the ORF of interest was grown in XY medium containing 2% raffinose and 0.1% glucose to an OD600 of 1.3 to 1.5. Expression was induced with 2% galactose for 1-1.5 hours, after which cells were centrifuged and washed in lysis buffer (LB: 50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2 or MgSO4, 50 mM β-glycerophosphate, 20 mM NaF, 2 mM benzamidine, 0.5% Triton X-100, 0.5 mM DTT, 10 μg/mL leupeptin, 2 μg/mL aprotinin, 0.2 mM AEBSF, 1 mg/mL pepstatin A). The cell pellet was resuspended in 1 mL LB per gram of cells and lysed by the glass bead method. Cell extracts were clarified by centrifugation at 14,000 rpm for 20 min in a microcentrifuge. Clarified extracts were incubated with 50-80 μL of anti-FLAG-sepharose resin (Sigma-Aldrich) for 1 h at 4° C., then washed three times with wash buffer (WB; 50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 50 mM β-glycerophosphate, 5% glycerol, 0.1% TritonX-100, 0.5 mM DTT, 0.2 mM AEBSF) and once with WB without Triton X-100. To help remove background proteins, beads were then incubated for 15 min at 4° C. (referred to as the pre-elution step) in HBS (100 mM Hepes, 100 mM NaCl, 0.2 mM AEBSF) with 100 μg/mL non-specific HA competitor peptide (YPYDVPDYA, SEQ ID NO: 6, Research Genetics). FLAG-tagged protein complexes were eluted twice for 10 min. at room temperature (referred to as the elution step) in HBS with 200 μg/mL FLAG peptide (DYKDDDDK, SEQ ID NO: 3, Sigma). Eluates and pre-eluates were precipitated with TCA/deoxycholate, washed with acetone, air-dried, resuspended in protein sample buffer and were separated by SDS-PAGE on a 10-20% gradient gel (Novex). Proteins were detected by colloidal Coomassie stain (Gel-Code, Pierce) and selected for band-cutting based on their specific presence in the FLAG-tagged complex.

[0232] Protocol B: YP2 bearing ptet-CFLAG constructs were grown to near saturation, diluted to an OD600 of 0.2 in DOB-Trp medium (QBIOgene) containing 2% glucose and 2 μg/mL doxycylin and then grown for a further 6-8 hours to a final OD600 of 1.2-1.5. Alternatively, BY4742 bearing pGAL1-CFLAG constructs were induced as above. Capture onto anti-FLAG resin was carried out as in protocol A with the following exceptions. Cells were lysed in buffer containing 50 mM Tris pH 7.3, 150 mM NaCl, 1 mM EDTA, 10 mM MgSO4, 50 mM β-glycerophosphate, 0.5% Triton X-100 and complete protease inhibitor cocktail (Roche). Pre-elution was carried out twice for 10 minutes at 4° C. in 50 mM Tris pH 7.3 with a mixture of Angiotensin (DDVYIHPFHL, SEQ ID NO: 7, Sigma-Aldrich) and Bradykinin (PPGFSPFR, SEQ ID NO: 8, Sigma-Aldrich) peptides at 50 μg/mL each or, alternatively, with 100 μg/mL of the peptide, YDDKDKD (Schafer-N, SEQ ID NO: 9). These peptides are quite efficient for the purpose of washing away non-specific binding polypeptides. FLAG-tagged protein complexes were eluted twice for 10 min. at room temperature in 50 mM Tris pH 7.3 with 200 μg/mL FLAG peptide (Schafer-N). All wash and elution steps were by gravity flow in 2 mL columns (Mobitech) and eluates were either precipitated with TCA as above or dried under vacuum.

[0233] Mass Spectrometry

[0234] Excised gel slices were reduced with DTT and alkylated with iodoacetamide essentially as described. In-gel digestion with porcine trypsin (Promega, Madison, Wis.) was carried out on an automated robotics system and the resulting peptides were extracted under basic and acidic conditions. Peptide mixtures were subjected to LC-MS/MS analysis on a Finnigan LCQ Deca® ion trap mass spectrometer (Thermo Finnigan, San Jose, Calif.) fitted with a Nanospray® source (MDS Proteomics), so that a much increased sample processing speed is achieved. Chromatographic separation was accomplished using a Famos® autosampler and an Ultimate® gradient system (LC Packings, San Francisco, Calif.) over Zorbax® SB-C18 reverse phase resin (Agilent, Wilmington, Del.) packed into 75 μM ID PicoFrit® columns (New Objective, Woburn, Mass.). A cluster of IBM NetFinity X330 computers were used to match MS/MS spectra against gene and protein sequence databases. Protein identifications were made from the resulting mass spectra using two commercially available search engines, Mascot® (Matrix Sciences, London, UK) and Sonar® (ProteoMetrics, Winnipeg, Canada). A relational database system called Piranha was developed to store and process raw mass spectrometric protein identifications. Overall, the sensitivity level that can be routinely achieved is about 50 fmol of protein loaded on to a gel. This benchmark takes into consideration all steps in the digestion/extraction/MS analysis protocol and not just specifically the MS portion.

[0235] A skilled artisan should readily understand that other equivalent instruments of similar function/specification, whether commercially available or user modified, can also be adapted for the purpose of practicing the instant invention.

[0236] Informatics Analysis of Data

[0237] The Finnigan LCQ spectrometers were set to analyze multiple samples at a high sample rate. When the bait protein was highly expressed, the cut band containing the bait which subsequently became the sample for the mass spectrometer contained very large amounts of bait protein. If a large amount of bait protein was present, then the protein may adhere to the column on the LCQ. The result was that the bait peptides on the column may “carry over” into subsequent samples for the mass spectrometer. This was the result of high mass spectrometer throughput coupled with high sensitivity. Steps were eventually taken to minimize or eliminate this phenomenon But in earlier data and in samples where it does appear, the “carry over” effect was accounted for as follows. Any bait protein that was identified within 10 samples (or more) following the last analyzed sample containing a bait protein was designated as “carry-over” and filtered from the data set.

[0238] When the immunoprecipitation eluates were loaded into wells on SDS-PAGE gels, eluates with very abundant amounts of bait protein on occasion would “spill over” into the adjacent lane. This spilled-over bait protein was at times identified by the mass spectrometer. If we identified a protein that was the same as a protein used as a bait on that gel and if it was loaded within 3 gel lanes on either side, we designated that protein as “spillover”, and it was filtered from the data set.

[0239] A portion of the data does not have the following proteins reported, even if they were identified by the mass spectrometer: Ssa1/2/3/4, Sse1/2, Tdh1/2/3, Asc1, Cdc19, Eft2, Eno1, Eno2, Fba1, Hsc82, Pgk1, Yef3, and ribosomal structural proteins. These proteins were found to bind promiscuously to many proteins. For a subset of the samples, these were not reported in the database for time considerations. The data is stored in its original state in the Sonar® database (ProteoMetrics, Winnipeg, Canada); the above proteins have not been excluded from the Sonar database.

[0240] Background Filtering Criteria

[0241] As a consequence of both the gentle isolation methods used to recover protein complexes from concentrated extracts and the ultra-sensitive mass spectroscopy used to identify proteins in each gel slice, we detected non-specific contaminants in each complex purification. These recurrent background species were filtered from the dataset according to the following criteria: (i) any protein found in association with 3% or more of the baits assayed; (ii) structural components of the ribosome, which were detected in virtually every preparation; (iii) all proteins that detectably bound to anti-FLAG resin in the absence of a FLAG-tagged bait protein (see Tables 4-6; excluded proteins listed in of frequency).

[0242] The Ty proteins are viral elements that are inserted in multiple places in the yeast genome. There is a distinct identifier for each one, even though they are all nearly the same (and generally indistinguishable by MS). It was decided that all Ty elements would be excluded from the filtered dataset due to their overall high frequency of identifications, even though any particular Ty protein ID may not have been reported many times. Table 6 lists all the different Ty proteins that were excluded.

[0243] One distinct advantage of the HMS-PCI approach is that non-specific interactions are more readily identified as the size of the dataset increases. An inherent difficulty with any data filtering scheme is that proteins that participate in many bona fide interactions are at risk of being excluded from analysis. Proteins of note in this category included actin, tubulin, karyopherins, chaperonins and heat shock proteins, all of which are known to form numerous distinct and biologically relevant complexes. As a specific example, many relevant interactions with replication factor A, an abundant trimeric complex involved in DNA replication and repair comprised of Rfa1, Rfa2 and Rfa3, were not included in the data set as a consequence of stringent filtering criteria (see Table 4). Application of these filtering criteria reduced the dataset to 4209 distinct protein identifications in association with 511 baits (Tables 2 and 3). In its entirety, the interaction set contains 1,841 different proteins or approximately 29% of the yeast proteome. Although, the filtering process eliminated 77% of the 18,411 putative interactions identified, it only eliminated 30% of the total unique proteins.

[0244] Filtering Proteins that Bound Just the FLAG Resin

[0245] To identify all the proteins that bound non-specifically just to the anti-FLAG resin, mock immunoprecipitations were done without the plasmid containing the FLAG-tagged protein. These were loaded on an SDS PAGE gel, and the entire lane was cut into band-size slices for analysis by mass spectrometry. This was done for both protocol A and protocol B. All the proteins found in these mock immunoprecipitations were used to exclude the same proteins identified in the data set as background. Mock immunoprecipitations done using protocol A were used to filter protocol A data, and mock protocol B immunoprecipitations were used to exclude protocol B data

[0246] Filtering of Promiscuous Binders

[0247] Proteins that bound to numerous bait proteins were excluded from the data set as promiscuous binders. Exclusion was based on the number of different bait proteins that a protein bound. A graph was drawn for the percentage of different bait proteins with which each identified protein associated (FIG. 5). The graph shows a distribution where above a certain percentage of baits bound by a protein, the percentage bound increases dramatically. This was then taken as the percentage of baits bound by a protein above which the protein is likely a background, promiscuous binder. The interacting proteins to the right of the dotted line in FIG. 5 were taken as background proteins because they bound many baits. This line corresponds to 3% of the total baits bound. The filter for protocol A and B was set such that any protein that bound 3% or more of the total of baits in the protocol A or B data set, respectively was filtered.

[0248] Filtering Immunoprecipitation Experiments

[0249] Immunoprecipitation experiments were excluded if any of the cut bands yielded 10 or more filtered protein identifications. These immunoprecipitations are likely technical errors that affected the “cleanliness” of the immunoprecipitation.

[0250] Analyses of Large-Scale Protein Interaction Datasets

[0251] To enable systematic comparisons of large-scale protein interaction data sets, it was necessary to develop models for representation of interaction networks. The HMS-PCI dataset was compared to two comprehensive high-throughput yeast two-hybrid (HTP-Y2H) datasets3,4 using interactions reported in the literature as a benchmark. An important consideration in such comparisons is that any given immunoprecipitation experiment reflects a population of protein complexes with unknown topologies, which cannot be accurately represented as pairwise protein interactions. Two models, spoke and matrix, were devised to represent these complexes as hypothetical pairwise interactions to allow comparison with HTP-Y2H pairwise protein interaction datasets. The spoke model represents the data as direct bait interactions with associated proteins as follows:

Complex: C={b, c, d, e}(b=bait; c, d, e=bait-associated proteins)

Spoke Model Interactions: is={b−c, b−d, b−e}

[0252] This model does not take into account indirect interactions between bait and the associated proteins (false positives) or interactions among the associated proteins themselves (false negatives). The matrix model represents the set of bait and associated proteins as an N×N matrix, with a row and a column for each protein in the set. All possible interactions between every protein in the set are then present in the matrix entries as follows:

Complex: C={b, c, d, e}

Matrix Model Interactions: iM={b−b, b−c, b−d, b−e, c−c, c−d, c−e, d−d, d−e, e−e}

[0253] This model takes into account indirect interactions and generates many false positives (false hypothetical interactions), but no false negatives (missed real interactions). Both the spoke and matrix representations of the HMS-PCI dataset follow a power-law distribution for connectivity (FIG. 4A)46-48.

[0254] All datasets were entered into the Biomolecular Interaction Network Database (BIND), which has been designed as a standardized repository for all forms of biological interaction data, including protein-protein and genetic interactions49. To systematically compile a set of published interactions as a benchmark, we used a search engine called PreBIND, a support vector machine (SVM) and natural language processing based algorithm used to help identify abstracts in PubMed that describe protein-protein interactions. Once a potential interaction is found by the SVM, it is vetted by an indexer and entered into BIND. Beginning with all bait proteins used in this study, PreBIND was used to collect a non-exhaustive set of 709 protein interactions from the literature. For comparison purposes, the HTP-Y2H and PreBIND datasets were normalized to correspond to baits used in this study. The spoke and matrix model representations of the HMS-PCI dataset contained approximately 3-fold greater published interactions than either of the HTP-Y2H studies (Table 3 and FIG. 4B, C). In particular, we detected 80 literature-validated interactions in the spoke model with 85 baits that failed to identify any interactions in the corresponding library-based HTP-Y2H screen3. Furthermore, over 148 common baits, an array-based HTP-Y2H screen4 yielded 29 validated interactions from 87 productive baits while the HMS-PCI approach generated 45 validated interactions from 121 productive baits. In addition to published interactions, a number of novel interactions were shared by the HMS-PCI and HTP-Y2H datasets (FIG. 4D).

[0255] It has been noted that the large-scale organization of metabolic networks in Archaea, Eubacteria and Eukaryotes are scale-free and follow a power law distribution for connectivity. Networks of this type are robust and error-tolerant. A similar power law distribution is also evident in HTP-Y2H interaction data sets. An analysis of the connectivity in the HTP-MS/MS network, in either the spoke or matrix representation, also revealed a power law distribution. Thus, the higher density of interactions in the HTP-MS/MS data set do not alter the overall properties of the network

[0256] Bioinformatics

[0257] All filtered interactions were entered into BIND, the Biomolecular Interaction Network Database. BIND is built around an ASN.1 specification standard that stores all relevant information about the interacting partners, including experimental evidence for the interaction, subcellular localization, biochemical function, associated cellular processes and links to the primary literature. BIND is an open source public database implemented by the Blueprint consortium and is freely available at the BIND web site. A BIND yeast import utility was developed to integrate data from SGD, RefSeq, Gene Registry, the list of essential genes from the yeast deletion consortium and GO terms. This tool ensures proper matching of any yeast gene or protein name to a protein coding region and accession number, and thereby eliminates nomenclature redundancy during import of yeast protein interaction data into BIND for visualization and analysis. Tools from the BIND project used here are written in ANSI C using the cross-platform NCBI Toolkit available at the NCBI web site. Programs were developed and run on the Linux and the Windows computer platform. Source code for the BIND database and data management system is freely available under the GNU Public License online. BIND records, tables of filtered and unfiltered protein complexes, and supplemental tables are available in electronic format at the MDS Proteomics web site.

[0258] For generation of hypothetical matrix interactions, a program called “spoke2matrix” was written to automatically convert protein complex data (i.e., the bait and associated proteins) to the matrix representation as described in the text. In instances where the same bait was used more than once, matrix interactions were generated from the results of individual immunoprecipitation experiments. A program called “common” was written to compare HMS-PCI and HTP-Y2H to literature-derived interactions detected with PreBIND. A program called “intfiltnorm” was used to normalize HMS-PCI and HTP-Y2H datasets to contain only interactions in which an interacting partner had been used as a bait in our HMS-PCI study. Interaction comparisons for overlap calculation purposes were treated as reflexive (i.e. A−B=B−A), and datasets were compiled as lists of pairwise gene names. All three programs described in this section convert an input list of yeast gene or protein name pairs to Refseq NCBI GI numbers for rapid internal processing using the BIND yeast import tool (see above).

[0259] Visualization of protein interaction networks was performed with Pajek, a program designed for large network analysis, and freely available for non-commercial use. BIND can export an arbitrary molecular interaction network as a Pajek network file. FIG. 4A was created with the Pajek program using a Fruchterman-Reingold automatic 3D layout with factor 3. Other network representations were manually constructed using Pajek. An additional program called “ip2fig” was written to create a Pajek network file with arrows pointing from bait protein to an experimentally determined associated protein and/or with previously known interactions from the PreBIND set highlighted.

[0260] The connectivity distribution of the spoke model network was calculated using the Pajek software package by partitioning the network by node (protein) degree (k). The resulting partition was exported to Microsoft Excel where the graph of the probability P(k) that a node in the network interacts with k other nodes was plotted versus k. The resulting graph could be fitted using a power-law with an R2 value of 0.92. The power-law relationship was P(k)=1098 k−1.7297. The fit of the connectivity distribution to this power-law was worse at higher values of k, most likely from the effects of the filter that was applied to the raw HMS-PCI data to remove background and from the fact that the spoke model does not take indirect interactions into account. Metabolic and protein interaction networks discovered so far follow a power-law connectivity distribution. Such networks are robust and maintain their integrity when subjected to random disruption of components. The distribution of the matrix model representation of the HMS-PCI dataset also followed a power-law relationship, but not as closely as the spoke model. The relationship was y=865.68×−1.2181 with an R2 value of 0.83.

[0261] The invention also uses standard laboratory techniques, including but are not limited to recombination-based molecular cloning, yeast cell culture, immunoprecipitation, SDS-PAGE electrophoresis, protein complex isolation, in-gel protease digestion, etc. Such information can be readily found in a number of standard laboratory manuals such as Current Protocols in Cell Biology (CD-ROM Edition, ed. by Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Joe B. Harford, and Kenneth M. Yamada, John Wiley & Sons, 1999).

[0262] Systematic Identification of Protein Interaction Networks in Saccharomyces cerevisiae by Mass Spectrometry

[0263] The recent deluge of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. Here, we report a large-scale application of mass spectrometry to identify protein-protein interactions in complexes isolated from the budding yeast S. cerevisiae. Beginning with over 10% of predicted yeast proteins as baits, more than 40,000 LC-MS/MS identifications of associated proteins were made. This raw data set was filtered to render a set of 4,209 detected interactions that covered 29% of the yeast proteome. Numerous inter-pathway connections and novel multi-protein complexes were identified in various DNA damage, cell cycle and signaling pathways. Compared to previous large-scale two-hybrid studies, we achieved a 3-fold higher success rate in detecting known interactions. High-throughput mass spectrometric approaches will permit comprehensive analysis of complex proteomes, including the set of all predicted human proteins.

[0264] Mass Spectrometry

[0265] As a preliminary survey of the yeast proteome, we chose a set of 725 bait proteins that represent a variety of different functional classes, including 86 proteins implicated in DNA damage and repair, 100 protein kinases and 168 baits used in array based two hybrid screens4. A small scale, one-step immunoaffinity purification based on the FLAG epitope tag was used to capture protein complexes. 1,362 individual immunoprecipitations were resolved by SDS-PAGE, followed by detection of specific proteins by colloidal Coomassie stain, excision of proteins from the gel and tryptic digestion for mass spectrometric analysis (FIG. 1).

[0266] Mass spectrometric identification of proteins is achieved by comparison of peptide mass fingerprints or partial sequence information derived from peptide fragmentation patterns to gene and protein databases8. Our isolation procedure often yielded complex protein mixtures from single excised bands, which could not be resolved by peptide-mass-fingerprinting alone. Therefore, we used MS/MS fragmentation to unambiguously identify proteins in each band. In yeast, as in higher eukaryotes, a single MS/MS spectrum of a unique peptide is often sufficient to identify a protein. To achieve high-throughput MS/MS protein complex identification (HMS-PCI), we constructed an automated proteomics network of mass spectrometers, based on nano-HPLC-electrospray ionization-MS/MS, capable of continuous operation. On average, we generated approximately 60 MS/MS spectra per gel slice that, when matched to the protein sequence database, allowed definitive identification of proteins even in complex mixtures. 15,683 gel slices were processed, yielding approximately 940,000 MS/MS spectra that matched sequences in the protein sequence database (Table 1). 40,527 protein identifications were made in total, corresponding to 18,411 potential interactions with the set of bait proteins (Table 1). An average of 3.1 proteins were identified per excised band. This raw dataset was filtered according to empirically derived criteria to yield 4,209 distinct proteins in association with 511 baits (Table 1). The filtered interaction set contains 1,841 different proteins representing 29% of the yeast proteome (Table 2; see also MDS Proteomics web site). Of the proteins identified, 734 corresponded to previously undocumented proteins predicted from the yeast genome sequence. Additional complexes identified subsequently are listed in Table 8.

[0267] Validation of HMS-PCI

[0268] The HMS-PCI approach was validated in part by detection of known complexes from a variety of subcellular compartments (Table 2). For example, we recovered all major components of the Arp2/3 complex that nucleates actin polymerization in the cytoplasm, including Arp2, Arp3, Arc15, Arc18, Arc19, Arc 35 and Arc409. Similarly, the eIF2 translation initiation complex, composed of Sui2/3, Gcd1/2/6/11 and Gcn3, was recovered with a Sui2 bait10. A number of transcription factor complexes were recovered, including the Met4 complex that regulates methionine biosynthesis gene expression. Notably, Met4 was detected in conjunction with the SCFMet30 ubiquitin ligase components Met30, Cdc53, Skp1, Hrt1 and Rub1, which negatively regulate Met4, as well as with its transcriptional co-regulator Met3111. We were similarly able to capture and identify multi-protein complexes in the vesicular (e.g., Vps21, Ypt1, Cop1), nucleolar (e.g., Nop13, Ygr103w) and membrane (e.g., Ras2, Yck1/2, Kin2, Kre6) compartments. Below we describe a limited subset of the numerous interactions detected by HMS-PCI, which illustrate the ability of this approach to discover protein function and to identify inter-pathway connections.

[0269] Phosphorylation-based Signaling Complexes

[0270] As protein phosphorylation underlies many cellular signaling events, the identification of biologically relevant substrates and regulators for kinases and phosphatases is crucial for a global understanding of cell regulation1. To approach this issue from a proteome-wide perspective, we used 100 of the 122 kinases encoded by the yeast genome, as well as 36 phosphatases and phosphatase regulatory subunits, as baits to capture associated signaling components (Table 2). As an example, we recovered numerous known and novel interactions with several mitogen activated protein kinases (MAPKs). In haploid cells, the mating pheromone/filamentous growth signal is transmitted by the archetypal MAPK module, Ste11/Ste7/Fus3/Kss1, in a response that has been under intense genetic and biochemical scrutiny for nearly 30 years12. HMS-PCI analysis of complexes captured with Kss1 identified many known components of the pathway, including Ste11, Ste7, and four known downstream targets, the transcriptional regulators, Ste12, Tec1, Dig1/Rst1, and Dig2/Rst2 (FIG. 2A, B). In addition, we identified other novel Kss1 interactions of potential biological significance. Bem3 is a GTPase activating protein that may be recruited to Kss1 signaling complexes in order to attenuate the Cdc42 Rho-type GTPase, an upstream activator of the pathway13. Bck2 is an activator of the G1/S transcriptional program that may be targeted by Kss1 during pheromone induced G1 arrest; indeed, a bck2 mutant is hypersensitive to mating pheromone, while overexpression of BCK2 causes pheromone resistance14. Biologically relevant interactions were also detected with other MAPKs, including between the cell wall integrity MAPK Slt2 and its upstream activators Bck1 and Mkk212, and between the osmotic stress response MAPK Hog1 and a downstream target kinase, Rck215. Consistent with its genetic role in attenuating the pheromone and cell wall integrity responses16,17, the dual specificity phosphatase Msg5 was associated with Fus3, Kss1 and Slt2 (Table 2).

[0271] Numerous proteins were detected in association with Cdc28, a cyclin dependent kinase that controls many aspects of cell division (FIG. 2C). We identified interactions between Cdc28 and its regulatory partners Cks1, an essential tight binding subunit, and the cyclins Cln1, Cln2, Clb2, Clb3 and Clb5 (ref. 18). Probable upstream and downstream connections to Cdc28 were also found. The dual-specificity kinase Swe1, which mediates the morphogenesis checkpoint arrest via inhibitory phosphorylation of Cdc28, was associated both with Clb2 and Hsl7, a known negative regulator of Swe1 (ref 19). A novel interaction between Swe1 and Kel1, a protein that is involved in cell fusion and cell polarity20, might signal the establishment of polarized growth to Swe1. Numerous events in mitosis are activated by Clb1/2-Cdc28, including a transcriptional positive feedback loop that controls expression of CLB1/2 and other G2/M regulated genes, via the forkhead transcription factors, Fkh1 and Fkh221. Cdc28 was detected in association with Fkh1, providing direct physical closure of the kinase-transcription factor circuit. In addition, Fkh1, Fkh2 and a related forkhead transcription factor Fhl1 were found in complex with one another. Fhl1 has not yet been implicated in G2/M transcriptional control, but given that a fkh1 fkh2 double mutant is viable, it is possible that Fhl1 contributes to transcriptional activation in the absence of Fkh1/2. Intriguingly, Fkh1 interacted with Net1, a nucleolar protein required for rDNA silencing and mitotic exit, and both Fhl1 and Net1 are required for proper Poll-dependent expression of rDNA genes22,23. Furthermore, both Fkh1 and Fkh2 associated with Sin3, a component of the histone deacetylase machinery that represses many genes24, consistent with the postulated role of Fkh1/2 as transcriptional repressors in other phases of the cell cycle21.

[0272] A recently discovered cell cycle pathway called the Mitotic Exit Network (MEN) is based on the protein kinases Cdc5, Cdc15, Dbf2 and Dbf20, the protein phosphatase Cdc14, and other proteins25. The polo domain-containing kinase Cdc5 was found in association with the cohesin complex, composed of Smc1, Smc3, Mcd1/Scc1 and Irr1 (Table 2). These interactions corroborate the recent finding that Cdc5 can phosphorylate the Mcd1/Scc1 subunit of cohesin to promote sister chromatid separation26. A novel interaction with the spindle pole body (SPB) protein Spc72 probably reflects localization of Cdc5 and other MEN components to the SPB in early M phase27,28. HMS-PCI also revealed connections between MEN components themselves, including Dbf2-Mob1, Dbf20-Mob1, Tem1-Bfa1, Tem1-Cdc15, as well as several novel interactions (Table 2).

[0273] Many protein kinases and phosphatases are regulated by tight binding subunits, which serve to localize or control activity1. We identified several known examples of interactions between kinases and inhibitory subunits, such as between the Tpk1/2/3 cAMP-dependent protein kinases and the regulatory subunit Bcy1, as well as between several cyclins and their cognate Cdk partners. The type 1 protein phosphatase catalytic subunit Glc7 regulates a variety of cellular processes by association with at least 6 different regulatory subunits, of which we identified 4 (Sds22, Reg1, Gip2, Glc8). Other novel interactions detected with Glc7 suggested a role in chromosome segregation and cell cycle (Cdc14, Ytm1, and Ygr103w), glycogen metabolism (Gph1), cell fusion and polarity (Kel1) and RNA processing (Fip1, Cft1 and Sen1). In other examples, we detected the regulatory subunits Cdc55, Rts1, Tpd3 and Tap42 in association with the PP2 phosphatases, Pph21 or Pph22. A protein of unknown function that is induced in response to DNA damage, Ygr161c, bound to both Pph21 and Pph22 and may represent a novel regulatory subunit. Another unknown, Ydr071c, interacted with the type PP2C phosphatases, Ptc3 and Ptc4. Taken together, the above examples demonstrate that HMS-PCI can readily chart protein complexes in phosphorylation-based signaling networks.

[0274] A Cellular Network—The DNA Damage Response

[0275] To test the ability of HMS-PCI to identify new connections and components in an entire biological process, we analyzed protein complexes centered on 86 proteins known to participate in the DNA Damage Response (DDR) in yeast. The DDR is critical for maintenance of genome stability and depends both on numerous DNA repair processes and on signaling cascades, called checkpoint pathways, that control cell cycle progression, transcription, apoptosis, protein degradation and the DNA repair pathways themselves29. The global DDR network revealed by HMS-PCI is not only highly enriched in known interactions but also contains many novel interactions of likely biological significance (FIG. 3). Examples of known interactions include: the replication factor C complex (RFC, Rfc1-5) and the RFCRad24 subcomplex, as well as the PCNA-like (PCNAL) Mec3/Rad17/Ddc1 complex, both of which transduce DNA damage signals; part of the Mms2/Ubc13/Rad18 post-replicative repair (PRR) complex; and the Mre11/Rad50/Xrs2 (MRX) complex that mediates double strand break repair by homologous and non-homologous mechanisms29. Although the small scale immunoprecipitations we used rarely yielded complete complexes, the comprehensive coverage of DDR proteins readily identified pathway and network connections. For example, we recovered Rfc4 in Ddc1 complexes, consistent with the hypothesis that the PCNAL complex might be loaded onto DNA by the RFCRad24 complex30. Our analysis of nucleotide excision repair (NER) proteins revealed the extensive network of interactions in this process (Table 2, FIG. 3). We recovered nearly all known nucleotide excision repair (NER) factors in their dedicated subcomplexes31: Rad1-Rad10-Rad14 (NEF1); Rad3-TFB3-Kin28-Ccl1 (NEF3/FFIIH) and Rad7-Rad16 (NEF4). The Rad4-Rad23 interaction (NEF2) was not found, but we nevertheless detected an association between Rad4 and NEF1, a known interaction among NER factors. In addition to these previously described interactions, the HMS-PCI approach unraveled novel interactions of interest in almost all aspects of the DDR, a few of which are presented below.

[0276] The Rad53 protein kinase is a central transducer of DNA damage29 and is the yeast orthologue of Chk2, the product of the gene mutated in the cancer syndrome variant Li-Fraumeni32. HMS-PCI analysis confirmed the known Rad53 interaction with Asfl33,34 and yielded several novel complexes of likely biological significance. Rad53 captured the PP2C-type phosphatase Ptc2, which is genetically implicated as a negative regulator of RAD53-dependent DNA damage signalling35. Furthermore, the uncharacterized gene product Ydr071c was detected with both Rad53 and the PP2C family members, Ptc3 and Ptc4, suggesting that Ydr071c may be a DDR-specific regulatory factor of PP2C-type phosphatases. Consistent with this physical interaction, we find a genetic interaction between YDR071C and RAD53 (R Woolstencroft and D. D., unpublished). With regard to Rad53 substrates, the putative targets Swi4 (ref. 36)and Cdc5 (ref 37) were directly or indirectly connected to Rad53 by HMS-PCI.

[0277] The Dun1 protein kinase has a similar overall structure to Rad53 and Chk2, most notably the presence of a phosphothreonine-binding module termed the FHA domain38. The HMS-PCI interaction profile of Dun1 included the potential upstream regulators Rad9, Rad53, Rad24, Hpr5 (Srs2) and Rad50. Of particular note is the interaction with Sml1, an inhibitor of ribonucleotide reductase that is phosphorylated in a DUN1-dependent manner, an event proposed to target Sml1 for degradation39. Dun1 also interacted with Rsp5, an E3 ubiquitin ligase reported to target the RNA polymerase II large subunit (Rpo21) for ubiquitin-mediated degradation following DNA damage40. Rsp5 is thus a candidate for the E3 enzyme that targets Sml1 for degradation after DNA damage.

[0278] Despite being one of the best understood DNA repair processes, some aspects of excision repair are still poorly defined. For example, the biochemical function of Met18/Mms19 has been particularly elusive31. The HMS-PCI approach revealed that Met18 can interact with Rad3, a component of the TFIIH complex needed for both RNA PolII-dependent transcription and NER A further regulatory connection is suggested by our detection of an association between Met18 and Bcy1, the regulatory subunit of the yeast cyclic AMP-dependent kinases. As deletion of BCY1 causes ultraviolet (UV) radiation resistance41, it is possible that Met18 links the PKA pathway to the NER machinery via its dual interaction with Bcy1 and TFIIH. Further links between excision repair and the ubiquitin system were revealed by analysis of Rad23, which contains a ubiquitin-like (UBL) domain, two ubiquitin-associated (UBA) domains and a unique region that binds Rad4 (ref. 31). The interaction detected between Rad23 and the ubiquitin chain assembly factor Ufd2 (ref. 42) is corroborated by genetic interactions that suggest RAD23 and UFD2 act antagonistically43. The Rad23-Ufd2 interaction may be mediated via the UBL domain since Ufd2 also interacted with another UBL-containing protein, Dsk2. We also identified an interaction between Rad1 and Msi1, a component of the yeast chromatin assembly complex44. Because deletion of MSI1 specifically causes UV sensitivity, the Msi1-Rad1 interaction suggests a means by which the chromatin assembly complex is recruited to UV-damaged DNA during NER.

[0279] Protein interaction data often suggests function, particularly when combined with protein sequence analysis. For example, we found that Rad7 interacts with the yeast elongin C homolog, Elc1, for which a function remains to be assigned. In mammalian cells, Elongin C associates with Elongin B, the cullin Cul2, the RING-H2 domain protein Rbx1 and any one of a number of substrate recruitment factors called SOCS-box proteins to form E3 enzyme complexes that mediate substrate ubiquitination45. Consistent with the Elc1-Rad7 interaction, sequence alignments revealed a divergent SOCS box motif in Rad7 (A. Willems and M. T., unpublished data). Rad7 may thus be part of an E3 enzyme complex that acts during excision repair.

[0280] Identification of Hypothetical Proteins

[0281] As a byproduct of HMS-PCI, we identified many proteins of unknown function whose existence had previously only been predicted from the genome sequence. Given the difficulty in prediction of coding regions from genome sequence information even in yeast, the direct identification of encoded peptides by mass spectrometry provides an important validation of putative coding regions. Table 7 contains a list of 734 proteins identified by mass spectrometry that fall into MIPS categories other than known proteins. Tables of hypothetical and putative proteins were obtained from the MIPS (Munich Information center for Protein Sequences) classification of ORFs from the MIPS web site.

[0282] Bioinformatics Elaboration of Protein Interactions

[0283] Even when unknown proteins do not fall within obvious large networks, protein interaction data often suggests function, particularly when combined with protein sequence analysis. For example, we found that Rad7 interacts with the yeast elongin C homolog, Elc1, for which a function remains to be assigned. In mammalian cells, Elongin C associates with Elongin B, the cullin Cul2, the RING-H2 domain protein Rbx1 and any one of a number of substrate recruitment factors called SOCS-box proteins to form E3 enzyme complexes that mediate substrate ubiquitination. Consistent with the Elc1-Rad7 interaction, sequence alignments revealed a divergent SOCS box motif in Rad7. Rad7 may thus part of an E3 enzyme complex that acts during excision repair.

[0284] In another example leveraged by bioinformatics analysis, we identified a hypothetical interaction network that contains an unusually large number of redox proteins associated with isoforms of Old Yellow Enzyme (OYE), Oye2 and Oye3. OYE was the first flavoenzyme purified, but despite extensive biochemical characterization of its NADPH oxidase activity, its true function is unknown. We identified 14 oxidoreductases of diverse functions in association with OYE isoforms, including Adh1, Rnr4, Sod1, Erg27 and Tyr1. An intriguing possibility is that OYE supplies oxidoreductase activity by channeling reducing equivalents to other oxidoreductases and their substrates, as mediated through specific protein-protein interactions.

[0285] Finally, it is likely that all protein complexes must be interconnected in order to allow coordination of diverse cellular functions. Such interactions should be readily revealed by non-directed, proteome-wide analysis. In one striking instance, we uncovered a large, previously undescribed network of interactions between proteins that are either localized to the nucleolus or involved in rRNA processing. One element of the network is formed by proteins of the U3 snoRNP complex, as revealed by interactions spanning several different baits. Similarly, the presence of several MEN proteins at the periphery of this network is consistent with the nucleolar sequestration of Cdc14 by Net1, and the role of Net1 in rDNA transcription. By virtue of their connections to the network, three proteins of unknown function, Ykr081c, Ylr427w and Yhr052w are implicated in nucleolar processing or regulation.

[0286] Prospects

[0287] The ultimate utility of any large scale platform rests upon its ability to reliably glean new insights into biological function. The instant invention provides the first high-throughput analysis of native protein complexes by highly sensitive mass spectrometric identification methods HMS-PCI. Importantly, proteome-wide analysis allows the detection of complex cellular networks that might otherwise elude more focused approaches. The numerous interconnections revealed in this study suggests that only a fraction of proteins need be investigated to obtain near complete coverage of the proteome. For example, linear extrapolation suggests that interactions captured with 2,500 bait proteins should connect the entire yeast proteome. Given that approximately 40% of yeast proteins are conserved through eukaryotic evolution50, the global yeast protein interaction map will provide a partial framework for understanding the human proteome. Imminent technical advances, such as the direct analysis of protein complexes without electrophoretic separation, as well as even higher sensitivity mass spectrometers, will undoubtedly extend the reach of the approach described here. Given that the set of proteins nominally encoded by the human genome is only 5-fold greater than the total number of yeast proteins, comprehensive analysis of the human proteome is feasible with current technology.

[0288] Methods

[0289] Recombination-based cloning, yeast culture and isolation of protein complexes were carried out using standard methods and are described above. Protein bands visualized by colloidal Coomassie stain were excised from polyacrylamide gels, reduced and S-alkylated, then subject to trypsin hydrolysis51,52. LC-MS/MS analysis was performed on a Finnigan LCQ Deca® ion trap mass spectrometer (Thermo Finnigan, San Jose, Calif.) fitted with a Nanospray® source (MDS Proteomics). Chromatographic separation was via a Famos® autosampler and an Ultimate® gradient system (LC Packings, San Francisco, Calif.) over Zorbax® SB-C18 reverse phase resin (Agilent, Wilmington, Del.) packed into 75 μM ID PicoFrit® columns (New Objective, Woburn, Mass.). Protein identifications were made from the resulting mass spectra using the commercially available search engines Mascot® (Matrix Sciences, London, UK), Sonar® (ProteoMetrics, Winnipeg, Canada) and Sequest® (ThermoFinnigan, San Jose, Calif.). Both the raw and filtered datasets generated in this study are available at the MDS Proteomics web site. The filtered dataset has been deposited in BIND49 and can be viewed at the BIND web site.

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[0359] All cited references, patents, publications are hereby incorporated by reference.

EQUIVALENTS

[0360] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

1TABLE 1Summary of HMS-PCI analysisBEFOREAFTERFILTERINGFILTERINGNUMBER OF 1368IMMUNOPRECIPITATONEXPERIMENTSNUMBER OF BAIT 724PROTEINS ATTEMPTEDNUMBER OF BAITS 605ASSAYED WHERE BAITPROTEIN WASIDENTIFIED BY MS1NUMBER OF BAITS 511ASSAYED WITH ATLEAST 1 COMPLEXINTERACTOR AFTERFILTERINGMS IDENTIFICATIONS42,275IDENTIFIED COMPLEX19,0854,171INTERACTIONS WITHTHE BAIT PROTEINUNIQUE PROTEINS IN 2,6041,821DATASET(42% OF(29% OFGENOME)GENOME)1proteins of less than 20 kDa were assumed to have run off SDS-PAGE gels.

[0361]

2

TABLE 2

Protein complexes detected by HMS-PCI.

BAIT
ASSOCIATED PROTEINS

AATI
MGM1, YJL204C

ABP1
ARP3, HXT7, MPS1, SCP1, SPS1, TUP1, YSC84

AFG3
ENP1, GCD6, IMD2, LPD1, MET3, YHR113W

AIP1
IMH1

AKL1
HTS1

APG12
AAC1, AAC3, ADE5, 7, APG17, ARC1, ARO1, ARP2,

CAR2, CPA2, CPR6, CRM1, CVT9, FET3, FET4,

GCD11, GFA1, IPP1, KAP122, KGD1, MET10, MET18,

PFK1, PPX1, PRB1, REP1, REX2, RPN1, RPN10, RPN11,

RPN3, RPN5, RPN6, RPN7, RPT1, RPT3, SEC18, TIF2,

TYR1, YDR214W, YGL245W, YHR020W, YHR033W,

YHR076W, YNL208W, YOR086C

APG5
CYS3, FET3, HST1, MOT1, PDR13, STI1, TSL1

APM1
APL2, APL4, BFR2, BRX1, CBR1, HEM15, KRE33,

KRE6, KRI1, KRR1, MDJ1, MGM101, NOG1, NOP4,

PAB1, PBP1, PWP1, RCL1, RPB5, RPN1, SEC28, SEC6,

SIK1, SNF4, THS1, TIF6, TUF1, UFD4, YBL104C,

YDR496C, YHR052W, YLR328W, YML076C, YNL294C,

YPK2

APM3
APL5, APL6, PLO2, SWI1, TRF5

APM4
BI2, CAF4, YJR072C

ARC40
ARC18, ARC19, ARC35, ARP2, ARP3, GCY1, NIP1,

NOP4, PDR13, POB3, RAD30, YLR241W, YNL040W

ARE2
PTC1

ARF1
ARF2, SHE10, YNL083W

ARL3
YKL206C

ARP2
ADE3, ADR1, ARC15, ARC18, ARC19, ARC35, ARC40,

AR07, ARP3, ATP3, BNA1, BNI1, CDC47, CDC54,

CIN1, DBF4, DED1, DUR1, 2, ECM17, FET3, GCD7,

GEA2, GFA1, GLG2, GSY1, HUL4, IMH1, KAP104,

KAP122, MET18, MSS18, NMD5, PDR13, PST2, PUF3,

RPN8, RVB1, SEC23, SEC26, STE5, TOM70, TRP3,

YGR016W, YJR029W, YKR065C, YMR018W,

YMR278W, YNL313C

ASC1
KRI1, LCP5, MSS116, PRP43, RFA1, SIK1, SIR3, SWI5,

YDL060W, YGR145W, YOR056C, ZIP1

BEM3
DOP1, YIL055C, YTA7

BFA1
FET4, KEX2, STE23, YGL121C

BMH1
ADR1, BNR1, BOI2, CSR2, CYK3, GSY2, KCS1, NTH1,

REG1, SOK1, STU1, SVL3, YFR017C, YIL028W

BMH2
CSR2

BRE1
YHR149C, YPL055C

BUB1
KAR4

BUB2
ISM1

BUD13
CLU1, KIP3

BUD20
ADH2, COF1, CPH1, GPI15, HHF1, HMO1, HTB1,

HTB2, HYP2, LSM2, MAM33, MDH1, MGM101, OYE2,

TEF4, YBL004W, YDR036C, YFL006W, YHR052W,

YIR003W, YLR004W, YPL013C, AFG2, FYV4, HHF1,

HTA1, HTB1, HTB2, KRE32, LHP1, MAM33, NMD3,

NOG1, NOP12, NOP13, PRP43, PUF6, PWP1, RSM24,

RSM25, YBL044W, YDR038C, YDR101C, YER006W,

YGL068W, YGR103W, YHR197W, YJL122W, YPL013C

BUD32
AAC3, CAR2, CPR6, CRM1, DIA4, GRX3, GRX4, HEF3,

IDP2, IMD2, IMD4, PHO81, POR1, RPN1, RPN5, RPN6,

RPT1, RPT3, SEC18, SEC23, URA7, YDR279W,

YHR033W, YJR072C, YKR038C, YML036W, YMR226C,

YOR073W

CAC2
RLF2, YDR453C, YLR080W

CAF20
CDC33, GAL83, NAP1

CAF4
ATP3, CCT2, CCT3, CCT5, CCT6, DPM1, ENT2, OSH7,

PRE2, SRP54, TCP1, YBL029W

CAR1
HYP2, IPP1, MDH1

CBF5
CRN1, MSS18, PAN5, SIK1, SRP1, VMA6, YIL104C,

YNL124W

CBK1
ARP2, ECM10, GAL7, MOB2, PRB1, SEC28, SGT2,

SIS1, SSD1, TAO3, UBP15, VMA6

CCE1
RNR3

CCR4
CDC36, CDC39, CYS4, POP2, RNQ1, RVB1, STI1,

UBR1, YGR086C

CCT2
ARC35, SEN2

CDC10
CDC11, CDC12, CDC3, IMD1, LPD1, SES1, SHS1,

TFG1, YPL191C

CDC11
CDC10, CDC12, CDC3, CLU1, PDI1, RPN1, TIF4631,

TIF4632, TOP2, YHR033W

CDC12
CDC11, CDC3, DOG1, DOG2, IMD4, MET6, MSK1,

PYC1, RGD1, SEC53, STB1, THI3, VMA22, YGL245W,

YKL056C

CDC13
BAT1, CPH1, ECM10, PRE6, SIP2

CDC14
AUK1, ATP3, ATP5, ATP7, DPM1, FUR1, GLC7, HEF3,

HMS1, MCR1, PDR15, SNF4, SPE3, TPS1, VAS1,

YDR453C

CDC15
AUT2, TFP1

CDC20
CCT2, CCT3, CCT5, MAD3, MDH1, MKK2, TCP1

CDC23
HYP2, SWM1

CDC28
CLU1, GSY1, MET10, RPN1, TCP1

CDC3
AAT2, ARG3, ARO4, CDC11, CDC12, HCH1, HYP2,

HYR1, NMD2, NTA1, TPD3, URA4, YBL032W,

YDR287W, YFR011C, YPL176C

CDC33
EAP1, FAA4, FRS2, GSY2, MKT1, RTT101, SLY1,

SNF4, TRP2, YDL239C, YDR214W

CDC4
SKP1

CDC42
ADH2, BEM4, CIK1, KCC4, SAN1, YBL032W,

YHL013C

CDC5
IRR1, KAP95, MCD1, NOP13, SMC1, SMC3, SPC72,

SRP1, YDR229W

CDC53
PDC6, POL30, POR1, PTC1, SKP1, YBR280C,

YLR352W

CDC55
CCT2, CCT3, CCT5, CCT6, GPD1, GSY1, HFI1, MSN4,

PDC6, PPE1, PPH21, PPH22, TCP1, TFG1, TPD3, YCK2,

YER077C, YHR033W

CDC7
BFR2, BIR1, ECM10, NUT1, PDC5, PDC6, PST2,

RPC19, SAR1, SEC27, STI1, THI3, TPS1, UBI4,

YLR231C, YLR331C, YLR386W

CDC9
DBP9, ECM10, POL30, YOR378W

CDH1
CCT2, CCT3, CDC28, CLB2, NA1, UBP15

CHK1
CTR1, GFA1, YLR152C

CIK1
CLU1

CKA1
CKA2, CKB1, CKB2, DBP10, DBP2, EGD1, ERB1,

HAS1, HHF1, HHT1, HOT1, HTA1, HTB2, KRE33,

KRI1, MGM101, NOG1, NOP12, NOP2, NOP4, NPI46,

PDI1, POB3, POL2, PUF6, PWP1, RRP5, SFP1, SIK1,

SPT16, SSF1, TIF4631, TIF6, TRL1, WTM2, YOD116C,

YER006W, YER084W, YGL104C, YGR090W,

YGR103W, YGR145W, YHL035C, YHR052W,

YKL082C, YLR002C, YPL110C, YRA1

CKA2
CKA1

CKS1
BUR2, CDC28, CLB2, CLB3, CLB5, CLN1, HYP2,

YDR170W-A, YER138C

CLB2
CDC28

CLN1
CDC2B, CKS1, PGM2

CLN2
ATP3, CDC28, ECM10

CMD1
CMK2, CMP2, COF1, CPH1, EDE1, HCH1, HUL5,

HYP2, ILS1, IPP1, MLC1, MYO2, MYO3, MYO4,

MYO5, NUF1, PGM2, PST2, SHE3, SHE4, SOD1, UBA1,

VAS1, VPS13, YNK1

CMK1
CMD1, VPH2

CMP2
CMD1, CNB1, IDH1, RFC3, RPN7, TEF4

CNA1
CMD1, YGR263C

CNB1
CMP2, CNA1, KRE6

CNM67
CAF4, FCP1

CNS1
ECM10, ILV5, YHB1

COF1
AIP1, CRN1, CYR1, GCN1, KAP114, PHO81, REX2,

SRV2, TOS3

COP1
ADR1, ATP3, CET1, HFI1, OSH1, PRP6, RET2, RGA1,

SEC21, SEC26, SEC27, SEC28, SPE3, TRP3, YBR270C,

YER140W, YJR072C, YLR405W, YPL222W

COQ7
COR1, IME4, PRP28, YJL068C

CPR6
ADH2, CAF120, QNS1, TRR1, YOR154W, YOR220W

CSE2
CDC33, POR1

CTF13
ARF1, SKP1

CTK1
CDC37, GBP2, HHF1, HRB1, KRE33, NPL3, SFP1, SIT4

CTK3
RVB1, STB3, UBA1, YBL032W

CYR1
CDC33, RNR2, SRV2

DBF2
CYR1, FAA1, GPH1, MOB1, RPN5, RPT3, RPT5,

SEC27, TFP1, TPS1, YJR072C

DBF20
ALA1, AXL1, EGD2, GPH1, IDH2, MOB1, RPB10

DBP8
CAR2, CDC15, CPA2, HEF3, KGD1, OYE2, PFK1,

PGM2, RNR1, RNR2, RPN1, SEC26, THI22, TIF2,

YDL086W

DDC1
MEC3, RFC4, SUV3

DIA2
BMS1, CDC46, CDC53, CKS1, COF1, CTF4, DBP10,

DED81, ENP1, ILS1, KRE33, KRI1, LST4, MCM2,

MCM3, NIP7, NMD3, NOP12, NPI46, PDR13, SEH1,

SKP1, SLT2, SPB1, SSF1, SSF2, TIF6, YAK1, YBL104C,

YHR052W, YJL109C, YKL014C, YPL012W

DIG2
ACO1, KSS1, SRP1

DMC1
ACC1, DPM1, HNM1, MDJ1, MES1, POR1, TRP3,

YDL148C, YDR516C, YLR106C

DPB11
NMD3, SRP1, TIF4631

DRC1
ADH2, ADH4, CKA1, COP1, GAL7, IPP1, MAM33,

MDH1, MGE1, MSU1, SRP1, TALl, TRL1, YHR074W

DSS4
AFG2, FAA4, NOG1, SEC4, YDR101C, YGR103W,

YJL122W, YPT1

DUN1
AAT2, ANC1, ASN2, DED81, PDX3, PRE8, VMA4,

YDR214W, YFL030W, YGR086C

DUR1, 2
BOI1

ELA1
EBP2, ECM10, ERB1, HAT1, IMD3, IMD4, KRE33,

LOC1, MSS116, NOG1, NOP1, NOP12, PET127, PUF6,

PWP1, TIS11, YAK1, YER077C, YGR086C, YGR090W,

YGR103W, YHR052W, YKR081C, YPL004C, YPL012W,

YRA1, YTM1

ELM1
TFP1

ELP2
ELP3, IK13, JIP1, ZMS1

ERB1
ACO1, CCT6, CDC14, EGD2, GND1, HAS1, HXT7,

MET6, MRT4, MUB1, NOG1, PRP43, SAH1, SCS2,

SEC53, SPB4, SSQ1, TIF6, UBR2, YER006W,

YGL111W, YGL245W, YLR002C, YOR206W, YTM1,

ARP2, BRX1, CRN1, EBP2, EXG1, FPR4, MRT4, MYO1,

NMD3, NOG1, NOP2, PIB2, RLP7, SCS2, TIF6,

YDR412W, YER002W, YGR103W, YHR052W,

YKR081C, YLR002C, YNL110C

ESS1
BCY1, CAR2, CPR6, HEF3, HSP104, HXT6, PUP2,

RPB3, RPN1, RPO21, SPT5, TFG1, TOM1, YGR090W,

YHR033W, YLR106C

EST1
CBF5, DBP7, HSH49, KRE33, MSS116, PDI1, PET56,

PUF6, PWP1, RRP1, YER077C, YJL109C, YKL014C,

YKR081C, YPL012W

FAA4
PSR2

FAP1
FPR1

FAR1
CLU1, COP1, RPT3, SRP1, SSK2, UBP15

FHL1
FKH1, FKH2, GCN3, HHF1, HMO1, HTA1

FKH1
CDC28, CEG1, CKA1, CKA2, CKB1, CKB2, FHL1,

FKH2, FYV8, GCD2, GCD7, GCN3, HHF1, HTB1,

MBP1, MGM101, MPH1, NET1, NOP1, RRP1, SEC2,

SIN3, SUI2, SUI3, UBP12, URE2, YGR017W, YMR144W

FKH2
ADH2, HTB2, INO80, SIN3

FPR1
AAT2, ADE3, ALA1, ASN2, CIN1, DED81, GDI1,

HOM3, HSH49, KRS1, LIP5, MLP2, MSK1, PDR13,

PET127, PRP28, THI3, THS1, URA1, YDR341C

FUM1
YHR113W

FUN11
CLU1, RPN1, TIF2

FUN31
GPH1, RVB1, YOL045W

GBP2
HPR1, IMD3, MFT1, RLR1, SUB2, THP2, YNL253W,

YRA1

GCD11
BNI1, CDC123, GCD1, SPT16, YDL172C, YNL091W

GCD2
GCD1, GCD6, GCD7, GCN3, PRP6

GCD7
FAA4, FET3, GCD1, GCD11, GCD2, GCD6, GCN3,

LOS1, MET18, MSH4, NMD5, PRO3, SAN1, SCW4,

SUI2, SUI3, VAC8, YAF9, YLR243W

GCN2
YNL213C

GCN3
BGL2, CBP6, CDC39, CRN1, DHH1, ENP1, FET3, FRS2,

GAL2, GCD1, GCD11, GCD2, GCD6, GCD7, GUF1,

HIG1, IMH1, ITR1, KGD1, LCB1, MAS6, MCX1,

MGM1, MKT1, NDI1, PET9, PRE2, PRE9, PRP16,

RPB11, SAN1, SCY1, SDH2, SDH4, SEC34, SLC1,

SPT15, TOM70, TRP2, TRP3, VPS8, YBR0140,

YGL101W, YHM2, YJR072C, YJR080C,

YKR046C, YKR065C, YOL101C, YPL207W

GCN5
ADA2, FET4, HFI1, SPT7, TAF60, TRA1, UBP8,

YCL010C

GDI1
SEC4, STE11, VPS21, YPT1, YPT10, YPT31, YPT32,

YPT52, YPT6, YPT7

GIP2
GDB1, GLC7, GPH1, GSY2, MDH1

GLC7
CFT1, CLU1, CYS3, ERB1, FIN1, FIP1, FPR4, GLC8,

GPH1, GSY1, GSY2, KEL1, MDH1, MHP1, NPI46,

PRC1, REG1, SCD5, SDS22, SEN1, SPB1, STI1, SUI2,

SUI3, TRR1, YAR014C, YDR412W, YFR003C,

YGL111W, YGR103W, YHR052W, YOR227W, YTM1

GLC8
GLC7, PHO85, PPZ2

GND1
HEF3, KIP3, MOH1

GPA2
GPA1, IDH1, PMA2, YGL245W, YMR029C

GRR1
CDC53, COF1, CPH1, FOL2, HTB2, PDC5, PDC6, PFK1,

POR1, SAH1, SKP1, UBI4

GSP1
DJP1, GSP2, HOM3, KAP95, MOG1, RHO1, RHA1,

SNF12, SRM1, YDL172C, YRB1

GYP6
TRS120, TRS130

HAL5
ITR2

HAP2
AAC3, APG17, ARP4, ATP3, CDC33, CIS1, CYS4,

FOL2, GRH1, HAP5, IPP1, KRE32, LOC1, MAM33,

NAP1, NMD5, POL5, PSE1, RHR2, SAH1, SAP190,

SPE3, SSK2, TIF2, TIF6, YER002W, YHR052W,

YKL214C, YNL063W, YPL166W, YPR085C, YRA1,

YTM1

HAP3
GSF2, SPT4, TFP1, YOR203W

HAT2
ARC35, BAS1, DIM1, GND1, HAT1, HIF1, YOR233C,

YPR105C

HEX3
PTP2, SSK1, SSK2

HIR1
YER066C-A

HOG1
RCK2, VID21

HPR1
MFT1, PRB1, RLR1, SSK2, SUB2, YDR214W

HPR5
DUN1, SEC23, SEC53

HRR25
AEP1, ACO1, ATP4, BUD14, CAR2, CDC25, CKA2,

COR1, CRZ1, CYS4, DCP1, DCP2, DNM1, EDE1, EGD2,

ENP1, GAS1, GCN3, GLC7, GPH1, HHF1, HSP104,

HXT7, HYP2, IPP1, LOC1, LTV1, MDH1, MDS3, MGE1,

NPI46, OYE2, PEX19, PIN4, PTC4, PUF3, RPC19,

RPM2, SAP185, SAP190, SAS10, SEC2, SEC23, SES1,

SFB3, SGM1, SIT4, TGL1, TSR1, VMA4, YBR225W,

YEL015W, YER006W, YER1380, YGL111W, YGR086C,

YKL056C, YNL207W, YOR215C, YPL004C

HRT1
ADR1, BBC1, CDC39, CDC53, CRM1, DUR1, 2, ECM29,

ECM33, FAA4, GAL3, GCN1, GUF1, HYP2, IDH1,

KIM3, MKT1, MYO2, PFK1, PMA2, RPA190, RPN1,

RPN8, RTT101, SEC27, TPS1, UBI4, VPS13, YAR009C,

YGP1, YLL034C, YLR035C-A, YLR106C

HSH49
CHD1, CPH1, MLC2, RSE1

HSP12
CPH1

HTA1
HHF1, HIR2, HTB1, KAP114, KRI1, NAP1, NOP4,

RET1, RPC82, RPO31, SPT16, YGR103W, YLR222C

HYM1
ADH2, FET3, KIC1, PEX19, UFD4

IME2
CCT2, TCP1

IMH1
BI2, ERG13

INO4
HHF1, HTB1, HTB2, MAM33, NIP7, NUD1, PSE1,

YDL001W, YDR324C, YGL099W

INP52
ADO1, POR1, RNQ1, SAP190, TIF2, YDR279W, YHB1

IST3
BUD13, CAR2, CPH1, DED81, PDR13, PGM2, SAH1,

YDR341C

ISU1
NFS1

ISW2
ISW1, KAP95

KAP104
AAC1, ABF2, ADR1, ALD2, ALD3, ARF2, ARP2,

AYR1, BGL2, CBP6, CKA2, COX2, DBP6, DED1, DIM1,

DOG2, DPS1, EMP47, ENP1, ERP1, GAR1, GSF2, GSP1,

GSP2, GTT1, HEM15, HFI1, HRP1, HTA2, ISA1, KEM1,

KTR3, MAK16, MAS6, MEP1, MLC1, MNN9, MNT3,

MRT4, NAB2, NDI1, NUP170, NUT1, QAC1, PAB1,

PCL8, PET9, PGM2, PMD1, PSD1, RHO1, RMT2,

RPC40, SAC1, SEC4, TFG2, TIM11, TOM20, TUS1,

WSP1, YBR270C, YDL063C, YDL113C, YDL114W,

YDL204W, YDR071C, YDR275W, YER182W,

YFL027C, YHM2, YKR046C, YNL035C,

YNR021W, YOR093C, YPL138C, YPR13SC, YRB1

KJN2
BUD14, CMP2, DOG1, GIS4, HSP104, KEL1, KEL2,

KRE6, POP2, TEF4, TFC4, UBA1

KIN28
MPS2, SCJ1

KIN82
YNR047W

KNS1
CAR2, TFP1, TPS1

KRE31
BRX1, BUD3, CCT2, CCT3, CCT6, CIN8, CLU1, HIR1,

HTB1, KRE33, NAN1, POL5, RVB1, SIK1, SSD1,

TIF4631, YGL068W, YGR090W, YJL109C, YKL056C,

YPL012W

KSP1
ARO1, BCK1, CHS1, CMP2, DBP7, PRI2, TPD3,

YHR186C, YNL201C

KSS1
ACO1, ARP7, BCK2, BEM3, CCT2, CYS4, DIG1, DIG2,

FET4, FUS3, GFA1, HAS1, HXT6, MKT1, MSE1, NAP1,

PHO84, PIM1, PMA1, PYC1, RPA135, RPN10, RPN8,

RVB1, SEN1, STE11, STE12, STE7, TEC1, UBI4,

YDR239C, YER093C, YGL245W, YHR033W, YJR072C,

YLR154C, YOL078W, YPR115W

LAP4
AMS1, BIK1, CPH1, DLD3, FUS2, GGA1, GLN1, HHT1,

HTB1, MPP10, SPO72, SUP45, UBP15, VMA4,

YDR131C, YFL034W, YNL045W, YOL082W

LAS17
AAC3, BZZ1, GAL2, HXT6, HXT7, MYO4, PEP1,

PHO84, RPN1, RPN12, RVS167, SLA1, SQT1, VMA6,

VRP1, YHM2, YNR065C

LCD1
ADH2, ADH5, HEM15, HHF1, ILV5, RNQ1

LEM3
GCN1, HXT7, KAP95, NMD5, TCP1, YHR199C,

YLR326W

LIF1
ANP1, CKA2, DNL4, MEC3

LIG4
ACO1, HTA1, KGD1, MAK16, NOP2, TIF6, YDR198C,

YGL111W, YGL146C, YGR103W, YHR052W,

YKR081C, YNL110C, YPL110C, YTM1

LSM2
ADE5, 7, DHH1, LSM1, LSM4, LSM7, LSMB, PAT1,

PRP24, RPN6

LSM4
PAT1, SEC26, TPS1, UBP15

LSM8
APA1, GAR1, LSM2, QCR2, RPN12, RPN8, RRP42,

SMB1, TIF6, YGL117W

LST8
YFR039C

LTP1
MOT1

LYS1
FOL2, POR1, TAL1

MAG1
AI1, FUN12, HHO1, HHT1, HTB2, IMD2, IMD3, IMD4,

MPH1, MSH2, NOP12, RET1, RPC82, TIF4631, T1F4632,

YGR090W

MAK11
ERB1, HUL5, NOP2, TIF6, YGR103W

MCD1
IRR1, SMC1, SMC3

MCK1
PNT1, TRM3, YIL105C

MDH2
EXG1, FAA4, IMH1, MET18, PDI1, RFC2, RPN5,

YIL108W, YMR093W

MEC1
AC01, CLU1, MDH1, STI1, YGL245W

MEC3
RAD17

MED4
NIT1, PEX6, ROM1, TOF1, YMR102C, ZRG17

MEK1
MSN2, NMD3, RPN1, TFG1, YMR323W

MET18
BCY1, PRB1, RAD3

MET30
CDC53, HRT1, MET31, MET4, RUB1, SIS1, SKP1,

TEF4, UBI4

MGT1
AHP1, ARF1, CPH1, DUN1, GND1, HEF3, HHT1, HTB1,

LHS1, MGE1, RIP1, SEC27, SOF1, UBR1, YDR214W,

YGL121C, YHR033W, YKL056C

MHP1
GLC7

MIG1
MSS116, NOP12

MIH1
C0R1, CPH1, HTB2, QCR2

MKK2
ARP2, BCK1, BUL1, IDH1, LYS12, PRB1, RGD1,

RNQ1, RPN1, RPN7, RVB1, YJR072C

MLH1
MGE1, YOR155C

MLH3
GCR2

MMS2
IRA2, RSP5, UBC13, YOR220W

MOB2
CBK1

MSG5
FUS3, KSS1, SLT2, TAL1

MSH1
MAS1, MAS2

MSH3
HTB1

MSH6
MSH2

MSI1
CRC1, RLF2, YKR029C

MSN5
GAL11

MUS81
ADH2, ANC1, CDC16, CDC33, CDC5, ERB1, HHF1,

HHO1, KRE33, KRI1, LOC1, MES1, MGM101, MKT1,

MMS4, NHP2, NOP12, NOP2, NP148, PWP1, RAD53,

RPC10, SEC23, UBI4, YER006W, YER078C, YGR090W,

YHB1, YKR081C, YMR226C, YRA1

NAN1
OND1, GND2, SAP1

NMR1
APG2, CDC55, FAT1, IFM1, SAP155, SIT4, SKT5,

TIM22, XRS2, YDL121C, VDR287W

NOP13
DBP7, DRS1, EBP2, IMD2, IMD3, KRI1, MSS116,

NOP4, PUF6, RRPS, TIF4631, YGRS03W, YHR052W,

YOR206W

NOP2
BRX1, CKB1, COX6, FET4, GAR1, KRE32, NIP7,

NMD3, NOP1, PHO84, RRP1, SIK1, YER006W,

YGL111W, YGR103W, YOR206W, YPL009C

NPR1
SIP2, UBP14

NTA1
ECM10, HSP104, IPP1, MDH1, MGE1, TFP1, VMA4

NTG1
ARP2, CLU1, ECM10, FET3, IDH1, PRB1, RFC2,

RPC40, TIF34, YDR214W

NUP84
NUP120, NUP145, NUP85, OPI3, SLU7

NUP85
CBP3, HEM15, NUP84, SEHI, YMR209C

OSH3
NOP4

PAC1
PH06, YPK2

PAC11
DYN2, EXG1, PTC4, YBL064C, YLR177W, YOR172W

PAC2
RPN1

PAT1
DCP2, DHH1, LSM1, LSM4, LSM7, PEX19, YGL121C

PBS2
FET4, NEP2, PTC1, SSK2

PCL6
PHO85

PCL9
COR1

PDS1
SRP1

PEP3
PTC1, SEC7

PEX7
BNI1, CCT2, CCT3, CCT5, CCT6, CYS3, ENT4, FZO1,

LAP4, LST8, MYO2, NEW1, PRI1, RPN6, SEC6, SEN2,

SIF2, UBR1, YFL042C, YIL077C, YKL018W

PFK2
PFK1, TIF2

PFS2
CCT2, CCT5, CCT8, CFTl, HGH1, TOP1

PHO85
AAC1, ADK1, CDC26, FZO1, GSP1, PCL10, PCL6,

PCL7, PHO81, RHC18, SRP68, TOM20, VMA5,

YDR214W, YDR453C, YER083C, YFL030W, YGR165W,

VHB1, YML059C, YNL127W

PHR1
MSD1

PIB1
UBI4

PKH1
TPK3, YGR088C, YPL004C

PKH2
HXT6, HXT7, YGR033C, YGR086C, YPL004C

POL30
CVS4, IMD4, MKK2, RPO31

POL4
RHO5

PPH21
HEM15, PPE1, PPH22, RPC40, RTS1, TAP42, TPD3,

YGR161C

PPH22
CDC55, HTA2, MKT1, PPE1, RPA135, RPB11, RTS1,

RVB1, TAP42, TPD3, YGL121C, YGR161C

PPH3
CCT2, CCT3, DIA4, STE12, TCP1, YBL046W,

YHR033W, YNL201C

PPS1
ADE13

PPZ2
GLC8, SDS22, YOR054C

PRE1
PRE10, PRE2, PRE3, PRE5, PRE6, PRE7, PRE8, PRE9,

PUP2, PUP3, SCL1, YHR033W, YKL206C, YLR199C

PRK1
ABP1, AKL1, ECM10

PRP11
ADH2, ADK1, CLU1, COP1, GPH1, NAN1, REX2,

SEC27, SHM2, SSK2, TEP1, THI22, TIF4631, UBP15,

YGR043C, YGR250C, YLR222C

PRP19
CEF1, CLF1, SNT309

PRP4
ARP2, CSE1, STI1, TOR1

PRP46
CCT5, CCT6, PFK1, SGT2

PRP6
AAT2, ADE13, ADE16, ADE3, ADE6, ALA1, APE2,

ARA1, ASN2, BAT1, BRR2, CLU1, CMD1, COX4,

CPR6, CYS3, DED81, DOT6, FRS1, GCY1, GLN1,

GPD2, GPH1, GUF1, HSL7, HYP2, ILV3, IMD3, KRS1,

LEU4, MAD1, MDH1, MDNH3, MES1, MMD1, MSH6,

MSK1, PAB1, PAC2, PDR13, PMI40, PRC1, PRO3,

PRP3, PRP31, PRP4, RRM3, RRP1, SAM4, SCC2,

SCP160, SIS1, TIF34, TRL1, TRR1, YMR099C,

YNL123W, YOR214C, YOR285W, YPL004C, ZTA1

PSO2
MGM101, YHR076W

PSR1
PHM7, WHI2, YSA1

PSR2
BRN1, BUL1, EXG1, HXT6, HXT7, SSL2, YOR352W

PTC1
TSL1

PTC3
COP1, ECM29, REP1, YDR071C, YGR205W, YOR086C

PTC4
GIN4, YDR071C, YDR247W

PTC5
PRS3, TIF6

PTP3
FET4, HHF1, RRP5

PWP1
BRX1, CCT2, CCT3, CCT5, CCT6, HEM15, TCP1,

YOL027C

PWP2
YDR449C, YGR210C, YLR222C

QRI8
AHP1, SIP2, SSK1, SSK2, TPK2, UBI4

RAD1
CAR2, DUN1, FAR1, GPD1, GPD2, MSI1, MSS18,

PDC8, PWP2, SEC6, SEN1, STE20, UBI4, YAL027W,

YDR324C, YGR086C, YHR033W, YLR368W,

YNL116W, YPL004C

RAD10
ARC1, CPH1, FUM1, PRO1, RAD1, RNR2, SAH1,

SOD2, TFP1, TIF2

RAD14
CCE1, CTF4, RAD1, RAD16, RAD4

RAD16
GND1, HHF1, HTB2, HTZ1, PDX1, RAD7, SHP1,

YDR453C, YMR226C

RAD2
PEX15

RAD24
CCT3, DUN1, RFC2, RFC3, RFC5, RPT3, TCP1,

YDR214W, YJR072C, YLR413W

RAD25
MKT1, ST11

RAD26
ACH1, ACO1, ADH4, BIO3, CDC33, ECM10, ERG20,

GDI1, MAM33, MDH1, QCR2, RAD3, RHR2, SEC53,

TEF4, TFP1, TIF2, YDR326C, YHR076W, YMR226C,

YMR318C

RAD27
POL30

RAD28
CCT2, CCT6, DUN1, TCP1

RAD3
AAC1, AAC3, ACO1, ATP3, CCL1, HOR2, HXT6, IDH2,

KIN28, LSC1, MDH1, MET18, RHR2, RPN1, RPN8,

RPT3, TFB3, TFP1, THI22, YBR184W

RAD30
GPH1

RAD50
DUN1, GPH1, MAM33, MKT1, MRE11, REX2, RPT3,

SEC27, SSK22, TFP1, VMA8, XRS2

RAD51
MLH1

RAD52
ALD5

RAD53
ASF1, CDC13, DUN1, EDE1, HTA2, IPP1, KAP95,

MDH1, PTC2, SMC3, SRP1, SWI4, TBF1, YDR071C,

YGR090W, YMR135C, YTA7

RAD54
MDH1, MGE1, YKL056C

RAD55
PTC3, YHR033W

RAD59
AAC3, ATP3, BEM2, ECM10, GCD11, HOM3, HOR2,

ILV2, NTG1, OPY1, OYE2, PGM1, PGM2, PRB1, PTC3,

RAD52, RHR2, RPB3, RPT3, SEC27, SEC53, TEF4,

UBA1, VMA8, YDR214W, YER138C, YGR086C, YPT31

RAD6
MED4, RAD18, UBR2, YGL057C, YMR251W

RAD7
ELC1, UBI4

RAD9
DUN1

RAS2
IRA1, RAS1, TSR1

RCK1
CBR1, FUS3, HOG1, IDH2, ROD1, RPN8, SNF1, SNF4,

YPR038W

RCK2
FET4, HOG1, VPS41

RED1
SEC7

RFA1
ACO1, ARP2, RPT2, RVB1, YER078C

RFA2
AHP1, CDC10, GCD11, HIR3, HTB1, MGM101

RFA3
AAC3, AR01, CYS4, HEF3, HOR2, HXT7, PGM2, RHR2,

YDR128W, YJR141W

RFC2
ACH1, ADE5, 7, ATP3, BRR2, CPA2, HEF3, PGM2,

RFC3, RFC4, ROM2, SRP1, VAC8

RFC3
MAP2, RFC4, RFC5, RNQ1, RPN11, RPT3, SHM2,

YCL042W, YMR226C

RFC4
ACO1, ADE5, 7, ADH2, EFO1, HSP104, RFC1, RFC2,

RNQ1, RPT3, SAN1, YDR214W, YGL245W, YHR020W

RHC18
HHF1, IMD1, IMD4, SRP1

RHO1
AAT2, ASN2, CLF1, DIA1, DLD3, FUM1, GIS1, GLY1,

ILV3, PST2, WTM1, YBL064C, YFR044C

RHO2
MER1, MKT1, POR1, RRP5, VPS21

RHO4
NMD3, PDR13, RPG1, URA1

RHO5
TRR1

RIM11
CDC25, CKI1, GCR2, GIN4, HOM3, IRA1, IRA2, MYO2,

MYO4, NAP1, PMD1, PRS2, PRS3, PRS5, TPS1, TSL1,

YDR170W-A, YER138C, YER160C, YJR027W,

YJR028W

RIM15
PHO13, PHO85

RIS1
APG7, NOP2, TIF6

RLF2
KAP95, SRP1

RNA1
CAR1, GSP1, GSP2, KGD1, YRB1

RNR3
ARP2, CYS4, HTB1, MAS1, MAS2, MKT1, RNQ1,

RNR1, RPN12, RPN9, YNL134C

RPA190
RPA135, RPA43, RPB5

RPC19
HHF1, RET1, RPA12, RPA135, RPA190, RPC40,

YFR011C

RPC40
ACC1, ACH1, ADE12, ADH2, ADK1, ADR1, ARF1,

ARF2, ARO4, BGL2, CDC60, DOP1, ECM29, FRS1,

GCD11, GFA1, GLT1, GLY1, GND1, GPD2, HTS1,

IDH2, ILV1, ISA2, KAP122, KRI1, KRS1, MDH1,

MET18, MGM1, NGL2, PAB1, POL30, PYC1, PYC2,

RET1, RPA135, RPA190, RPA49, RPB5, RPC19, RPC25,

RPC34, RPC82, RPN3, RPO26, RPO31, RVS167, SEC27,

SMC4, SRY1, SSQ1, TBS1, TFP1, THS1, TOM40, URE2,

VMA4, VMA5, XRS2, YDR214W, YDR453C, YER138C,

YFL042C, YGL248W, YGR086C, YHR112C, YPL004C,

ZUO1

RPL5
MLP1, RLP7, TIF6, YHR052W

RPN5
EMP24, KGD1, KRE6, RPN1, RPN12, RPN6, RPN8,

RPN9, RPT1, RPT2

RPP0
AHP1, HHF1, HXT7, HYP2, NMD3, TIF6, YER067W,

YGL068W, YHR087W, YLR287C

RPT3
ARP2, CPR6, HYP2, IDH2, LHS1, MKT1, NAS6, POR1,

RPN1, RPN10, RPN11, RPN12, RPN3, RPN5, RPN6,

RPN7, RPN8, RPN9, RPT1, RPT2, RPT4, RPT5, STI1,

UBC12, YGL004C, YLR106C

RRP9
CBF2, DYN1, JEM1, NET1, NOP13, NOP13, PRO1,

YBL004W, YGL146C, YLR211C, YOL078W

RSP5
BUL1, DUN1, HXT6, PHO84, RNQ1, RPB3, RPB5,

RPO21, RPO26, YGR136W, YKR018C, YLR392C

RTF1
SF17, YHR009C

RVB2
RVB1

RVS161
ARG4, CRN1, DLD3, HSM3, MGE1, POR1, RVS167,

YGL060W, YOR118W

RVS167
COR1, DBP5, DBP9, DED1, ECM29, FRS1, FRS2,

FUM1, GIP2, GPD1, HOM6, HYP2, IDH1, ILV5, KRS1,

LPD1, LYS12, MAM33, MET18, NDI1, PDX3, PHO84,

PMI40, PRE10, PRE9, RGA1, RNA1, RSP5, RVS161,

SEC6, SER33, SES1, UBI4, UBP6, UBP7, URA7,

YBL036C, YER138C, YHR022C, YLR243W, YPL249C,

YSA1

SAC6
CNM67, LPD1, MDH1, SLF1, TRR1, XRS2, YER147C,

YKL075C

SAL6
SDS22

SAN1
ARP2, CDC54, RPA135, SRP1, UBI4, YPL113C

SAPI55
FLR1, SAC1, SDF1, SIT4, TIM22, YDL113C, YLR222C

SAP185
ANC1, ARG4, ARP4, ATE1, CDC33, CKA2, DUR1, 2,

EPL1, ESA1, GSY1, HRR25, MPT1, PET9, POR1, PSD1,

SDF1, YGR002C, YHM2, YMR209C, YPR040W, YRA1

SAT4
PHO85

SDS22
FYV14, GLC7, HXT6, NET1, NSR1, PMA1, PMA2,

PPZ2, REG1, RSE1, RVB1, SNF4, YGR130C, YHR186C

SEC13
NUP133, SEC31, YHL03PV

SEC27
ARG4, ARG5, 6, AYR1, BIM1, BTN2, CCT2, CCT6,

COP1, COR1, CPR6, CTR1, DNH1, EAP1, ERG27,

FAA4, GAL7, GIC2, HFI1, IDH1, IML2, KAP122, MAE1,

OM45, PCT1, PET9, PRB1, PRE10, PRO3, PTC3, RET2,

RPN7, RPT3, RVS161, SEC18, SEC21, SEC26, SEC28,

SEN54, STI1, TCP1, TIF34, TIF35, YBR187W,

YCR076C, YDL204W, YER049W, YGR086C, YGR235C,

YHR209W, YKR007W, YKR046C, YKR067W,

YNL181W, YNR021W, YOR051C

SEC31
CRN1, IDP3, SEC13

SEH1
ADE13, APE3, MYO1, NUP145, NUP84, NUP85, SEC13,

SUB2

SEN15
AAT2, ACH1, ACO1, AFR1, AHP1, ARC1, ARF1, ATP3,

CAR2, CDC33, CLU1, COF1, COR1, CPH1, CYR1,

CYS4, EGD1, ERG13, ERG6, FPR1, FRS2, GND1,

GND2, GRX1, HEF3, HHF1, LRO1, MET6, NTF2, OYE2,

PFK1, PRM2, RNR2, RSN1, SAH1, SCP160, SEC53,

SES1, SNU13, SOD1, TEF4, THS1, TIF2, UBA1, VMA4,

VMA5, WTM1, VBR025C, YDR453C, YGL245W,

YGR086C, YKL056C, YNK1, YPL004C

SET1
BRE2

SFP1
LAS1, MRS6, RNQ1

SGN1
CLU1, FUN12, NPL3, PDI1, PUB1, SPT2, TIF4631,

TIF4632, YGR250C

SHE2
KTR3

SHE3
MLC1, MYO4, SUL2, SUP45

SHS1
ACC1, ARC35, ARP2, ATP3, BGL2, DIM1, GSY1, HIS4,

MET3, MKT1, PUP2, RNQ1, RPB3, RVS167, SDH2,

YHR033W

SIF2
OSH2, TFP1, TRM3, VID28, YCR033W, YEL064C,

YIL112W, YLR409C, YMR155W, YRF1-3, ZDS2

SIP2
ARC35, GAL83, IDH2, SEC53, SNF1, SNF4, TCP1

SIR3
COR1, CYS4, GAS1, ILV5, RNR2, SAH1, SES1, SIR1,

TEF4, TFP1, TFP1, UBP8, YMR226C, YMR318C

SIR4
BLM3, SEC53, SIR2, SIR3, SRP1, YFL006W

SIT4
ACC1, ALG2, ARP2, ARP3, ATP3, BGL2, CCT6,

CDC42, CDC47, CHL4, DED1, EXG1, FAA4, GAD1,

GLT1, GSF2, HFI1, HXT3, HXT5, ILV1, MAE1, MSS18,

PPH3, PRE1, PRE6, PRE9, RMT2, RPB3, SAP155,

SAP185, SAP190, SCW4, TAP42, TIM22, WBP1,

YDL204W, YDR380W, YGR161C, YHB1, YNR033W,

YJR072C, YMR196W, YPR090W, ZRC1, ZWF1

SIW14
HXT6, YDR516C

SKI8
AKL1, SKI2, SKI3

SKM1
HMG2, PTC1, TPD3

SKP1
BOP2, COC4, CDC53, PRB1, SGT1, UFO1, YDR131C

SKS1
PRP28

SLN1
COP1, GCN3, LRS4, MDM1, VHR197W, ZRC1

SLT2
ARP2, BCK1, CPR6, EGD2, FOL2, GAL7, GND1, IDH1,

ILV5, IPP1, KIC1, KIN2, LHS1, LYS12, MKK2, MKT1,

OYE2, PDC6, PMA1, QCR2, RPN6, RPT3, SIS1, SMK1,

TIF2, YDR214W, YGR086C, YLR187W, YOR220W

SMC1
SMC3

SMK1
BUD7, COR1, GAL7, MAE1, PRE3, QCR2, RNR2, SLT2,

STI1

SML1
AAC3, ADH3, ATP3, DUN1, ECM10, GPH1, HIR3,

HOR2, NAT1, PFK1, PYC1, RNQ1

SMT3
CPH1

SNF1
ARF1, GAL83, GIS4, PRB1, SEC7, SIP2, SNF4, UBI4,

YMR086W

SNF4
GPH1, PST2, ROD1, SIP1, YOR287C

SNP1
BCV1, COR1, DOG1, ENP1, FET4, HAS1, MAM33,

NPI46, PIM1, PRP8, QCR2, SAP185, SAP190, SIT4,

SRP1, YLR386W

SOF1
CCT2, CCT3, CCT5, CCT6, KRE33, RRP5, TCP1

SPC24
BGL2, GCD11, GLT1, GPH1, ILV1, KAP122, MET18,

NRG2, SPC25, TID3, TIM13, YER182W, YHR182W,

YMR018W

SPC25
CTF18, SPC24, YLR381W

SPO12
PSE1, SRV2, SUM1

SPO13
IDH2, TIF2

SPS1
ARP2, ATP3, CPR6, IDH1, NMD5, PHO84, PPH21,

PPH22, PRB1, REP1, RPN8, SDH2, VMA8, YDR214W,

YDR372C, YHR033W, YKR046C

SPT2
AAC3, CKA1, CKA2, CKB1, CYS4, GND1, GSP1,

IMD2, KRE31, NOP1, NOP12, PUF6, RLI1, SAH1, SRP1,

SSF1, STE23, SUP45, TIF4631, YGR090W, YKR081C

SPT8
YML002W

SRP1
BLM3, CNA1, CPR6, DIS3, EAF3, FIP1, FYV14, HAS1,

HPR1, KAP95, MES1, MFT1, NAM8, NHX1, NUP1,

NUP2, NUP60, PAP1, PCT1, PDS1, REB1, RLR1, RNT1,

RRP4, RRP43, RRP6, RTT103, SIF2, SIN3, SNU56,

STO1, TRA1, UME1, YPR090W

SSK1
EST1, SSK2, SSK22

SSK2
DED81, DJP1, DPM1, GLT1, ILV1, LSC1, PTC3,

TOM70, YCG1, YDL113C, YLR154C, YNL051W

STE4
ADH2, ARP2, ASN2, CCT2, CCT3, CCT5, CCT6,

GCD11, GPA1, LAP3, PDC6, RNQ1, SUI2, TCP1, THS1,

VMA5, YDR214W, YHR033W

SUI1
AAT2, ALA1, APE3, ARG4, ASN2, CDC60, COF1,

DED81, ENP1, FUM1, HCH1, HYP2, MET14, NAS6,

NIP1, PDC6, PDR13, PRP9, RPG1, RPO21, RPO31,

SAR1, SAS4, SPT6, SUP45, TIF34, TIF35, TRR1, URA1,

VID28, YGR169C, YKL056C, YOR177C, YPL067C

SUI2
CDC33, FAL1, GCD1, GCD11, GCD2, GCD6, GCN3,

RFA1, SPT16, SUI3, TIF2, TIF4631, T1F4632, VPS4,

YBL032W, YLR400W

SWE1
AHC1, CLB2, COP1, HSL7, KEL1, UBP15

SWI5
ARP4, FAA2, HFI1, RPO31, SPT7, STB4, TRA1,

YGR002C

SWM1
ARO1, ARP2, CPA1, PRB1, PRP28, RNQ1, URA7,

YML072C

SXM1
ECM1, LHP1

TAF90
CCT2, CCT3, CCT5, NTG2, RSC1, TCP1, YDR287W,

YER160C, YJR072C, YNR065C

TEC1
HHF1, HTB1, STE12

TEL1
YPL110C

TEM1
ADK1, BFA1, CDC15, CDC33, CLU1, COF1, COR1,

CPH1, CPR3, CYS4, DUT1, EFB1, FAA4, GCD11,

HAS1, KGD1, LAP4, MCX1, NMD3, NUP53, PFK1,

PST2, RNR1, RNR2, RVB1, SAR1, SEC53, SSD1,

TEF4, TIF2, UFD4, VMA5, YER281C, YGL245W,

YGR066C, YHB1, YHR033W, YMR226C, YNK1, YTM1

TEP1
ILV2, MLH3, MLP2

TFB3
RAD3

TIF2
CAC2, CDC33, MED4, MLP1, MSK1, NDJ1, RAD5,

ROM2, TFG1, TIF4831, TlF4632, YJL107C

T1F34
RPG1

TOM1
PDR15, PRP6

TOP1
ACO1, CLU1, RPC82, SPT16, YFR011C

TOP2
CKA2, CKB1, DUN1, SIK1, YLR154C, YRA1

TOS3
SNF4, YKR096W

TPK1
BCY1, FET4, RIM15, TCP1, TPK2, TPK3, VAC8, VPS13

TPK2
BCY1, ECM7, MST1, SEC28, TID3, TPK1, TPK3,

YIL005W, YJR054W

TPK3
ADE5, 7, BCY1, CPR6, GPH1, PFK1, SEC27, TPK1,

YHR033W, YHR214W-A, YNL227C, YPT7

TPT1
KRE33, NOP1, YKR081C

TRF4
IMD1, IMD3, IMD4, MTR4, NAP1, PSE1, SIK1,

YDL175C, YIL079C, YPL146C

TUP1
APG16, CDC42, CLU1, COS7, CYC8, ECM10, GPH1,

NFS1, PET127, RLM1, SEC27, SPH1, SSY5, VID22,

YHC1, YHR052W, YIL082W, YKL116C

UBA1
FOL2, SER1, STI1

UBC1
ADK1

UBC12
ULAl

UBC13
AR09, MMS2, RAD18, REX2, UBA1

UBC4
QCR7, UFD4

UBC6
ATP4, GCN1, LOS1, POL5, RVB1, SEC7, UBA1,

YBL004W, YKL056C, YPT1

UFD2
DSK2, HMF1, NPL4, RAD23, SHP1, TSL1, UBI4,

YDR049W

ULA1
PPH22

UME1
MSH3, MSS116, RPD3, RRP5, SIN3, YBL004W,

YKR020W, YOL114C, YPL158C, YPL181W, ZRT1

URA3
RNQ1

VAN1
CBR1, COX2, DPM1, HOC1, ISW2, KTR3, MNN9,

NAP1, SCC3, SCJ1, SLC1, SPT15, WBP1, YJR072C,

YLR243W, YTA12

VPS21
ARO3, CDC60, GDI1, GPX1, IMD2, MRS6, STE11,

YML128C, YPT1, YPT52, YPT53

VPS41
PDR15, PEP3, PRP4

VPS8
MPS2, TFG1

WHI2
CSR2, HYP2

WTM1
CCT2, CCT3, CCT6, TOP1, WTM2

WTM2
CCT6, KAP104, MSS1, RNR2, RVB1, TSL1, WTM1,

YJL069C, YOR283W

XRS2
AHP1, CDC16, ERG20, MRE11, PST2, RAD50,

YBR063C

YAK1
AHP1, CDC39, DNM1, GDB1, RAD50, UBP15, UBR1,

VPS1, YDR453C, YLR241W, YLR270W, YOR173W,

YPL247C

YAR003W
RNR2, UB14

YBL036C
ADK1, FET4, HXT6, HXT7, PDC6, SES1, YOL078W

YBL049W
CCT2, CCT3, FYV10, VID26, VID30, YCL039W,

YDR255C, YMR135C

YER094W
CKA2

YBR175W
HPR1, RPN1, SET1, SFP1, SGS1, SUV3, YOL045W

YBR203W
NAP1, SKP1

YBR223C
RNQ1, SHP1

YBR267W
YJL122W

YBR280C
AAH1, CDC53, PRB1, YBR139W

YCK1
AAC3, ADH2, AHP1, APC1, BCY1, CAR2, CDC4,

CYS4, FOL2, GND1, HYP2, ILV5, LYS1, MPC54, OYE2,

OYE3, POR1, PPH21, PPH22, PST2, PYC2, RGR1,

RLR1, SAH1, SIP2, SNO2, SOD1, SSN8, THI22, TIF2,

TPD3, TPK2, TPK3, UBA1, VPS21, YBL108W,

YBR028C, YCK2, YCK3, YGR111W, YGR154C,

YHR112C, YJL207C, YMR226C, YPT53

YCK2
YCK1

YCL039W
BUD5, CTF19, FUN14, FYV10, HXT7, MNN1, PXA1,

SES1, SIF2, TFP1, UME1, VID24, VID28, VID30,

YBLO32W, YBL049W, YDR255C, YIL097W,

YIR020W-B, YMR135C, YOL087C, YPL1330

YCR001W
RAD23

YCR079W
CDC60, FAA1, HEF3, KGD1, MDH1, PRO2, PYC1,

RAD1, TIF2, TPS1, VID31, YPL110C

YDL025C
CDC33, YAL049C, YGR016W, YHR009C

YDL060W
HTB1, NOP12, YER006W, YOR056C

YDL100C
DLD1, GSF2, LAP4, MNN1, MSN4, POR1, YBR014C,

YER083C, YGL020C, YGRO86C, YLR154C

YDL156W
CCT2, CCT3

YDL175C
ADH2, DED1, NPL3, QCR2, SES1, SRP1, YGR165W

YDL193W
GCN1, GSP1, GSP2, NMD5, PMA1

YDL213C
CBF5, CBP2, CDC33, DBP7, DRS1, ERB1, GBP2, HAS1,

HMO1, HTB1, HTB2, IMD1, IMD3, ISA1, KAP95,

KRE33, KRI1, KRR1, MGM101, MSS116, NOP12, NOP2,

NOP58, NPL3, PET127, POL5, PRP43, PUF6, PWP1,

RLI1, RRP5, TIF2, TIF4631, TIF6, TRA1, TSR1,

YBL004W, YER006W, YGL068W, YGR103W,

YGR145W, YGR150C, YGR198W, YHR052W,

YJL109C, YJR041C, YKL014C, YKR081C,

YOR206W, YPL012W, YRA1, YTM1

YDR128W
CCT6, CPR6, DIM1, FAR1, GSF2, GSY1, GUF1, MDJ1,

NGG1, NPR2, POX1, RMT2, RNQ1, SEC6, SEH1,

VMA6, YDL113C, YDR2330, YER182W, YHR033W,

YJR072C, YNR018W, YPL207W

YDR131C
SKP1, YRB2

YDR165W
CDC53

YDR200C
FET4, PHO84, YFR008W, YGR066C, YMR029C,

YPL004C

YDR219C
SKP1, YHR122W

YDR247W
MER1, NUM1, PTC4, SEF1, SKT5, SPT16, SYF1, TPS1,

YDR071C

Y0R266C
CLU1, MGE1

YDR267C
ANC1, DOG1, MET18, RPN8, UBP9, YBR030W,

YLR349W, YLR392C, YOL111C, YOR164C, YPL068C

YDR306C
CDC53, MDH1, PGM2, SAH1, SKP1, SRV2, STI1

YDR316W
BUD9, DAK2, THI22, VMA6, YBL104C

YDR339C
COR1, PMC1

YDR365C
CDC33, CKA1, CKA2, CKB1, HTB1, IMD3, LHP1,

MSS116, NOP12, PMA1, YCR087W, YDR102C,

YJL207C, YKR081C, YNR054C, YRA1

YDR398W
ACC1, CPR6, CES1, ECM8, FAA4, GUF1, RMT2,

SEC28, SEC6, SGD1, YER138C, YGR210C

YDR482C
TPD3

YER041W
FPR4, POL30, YKR081C

YER066C-A
NMD2, PEX19, STI1, TOM70, YBL049W

YER117W
BCP1, FET4, IMD4, YPL208W

YFL034W
YPL110C

YFR003C
GLC7, MGE1

YFR016C
CAP1, CAP2, COF1, KOG1

YFR024C-A
ARO1, CKB2, PRP12, UBP15, YFR024C, YJL045W,

YLR422W, YOR042W

YGL004C
HSM3, NAS6, RPC40, RPN1, RPN10, RPN11, RPN13,

RPN3, RPN5, RPN6, RPN7, RPN8, RPN9, RPT1, RPT2,

RPT3, RPT4, RPT5, YKL195W

YGL081W
COP1, CYS4, GFA1, GSY1, NIP1, RFC4, SMC3, UBR1,

URA7, YER006W

YGL131C
YLR413W

YGL220W
GRX3, GRX4, YLL029W

YGR052W
APC2, ARF2, HIS4

YGR054W
HTB2, KRE33, NPL3

YGR067C
CLU1, HTB2, MKT1, SCP160

YGR103W
CKA2, CKB2, DBP10, HAS1, MAM33, NOP1, RRP1,

RRP5, SPB1, SRP1, TIF6, YER006W, YKR081C,

YPR143W, YTM1

YGR173W
MOH1, YDR152W

YGR223C
ERG10

YGR280C
PRP12, YPL110C

YHL010C
NHA1, YBL049W, YKR017C

YHR052W
EBP2, ERB1, KRE33, MAK5, MSS116, NOP2, NOP56,

RRP5, YOR206W

YHR105W
VPS13

YHR115C
YNL116W, YNL311C

YHR186C
VPH2

YHR188C
ARF1, ARF2

YHR196W
GND1, GPH1, HSP104, KGD1, NAN1, PFK1, SCS2,

TPS2, YJL109C

YHR197W
ATP3, BUD3, HTB2, RPC19, YDR131C, YNL182C

YHR199C
AEP1, IFM1, PSE1, TRX2

YIL007C
AC01

YIL079C
DED1, HRB1, IMD4, NPL3, TRF4

YIL113W
SLT2, SRV2

YJL020C
CPH1, GSY2, HTB2

YJL068C
TAL1

YJL069C
CKA1, CKA2, CKB1, CKB2, DIP2, KRE33, LAS1, LCP5,

NAN1, NGG1, NOP1, PRP40, PTC5, PWP2, RRP5, SIK1,

TFP1, YDR449C, YGR090W, YJL109C, YML093C,

YKR060W, YKR096W, YLR222C, YLR409C,

YML093W, YOR1450

YJL149W
CDC53, SKP1

YJR061W
KKQ8

YJR110W
RGR1

YJU2
CCT5, COR1, DED81, DUN1, EGD1, GCD11, NAP1,

NMD3, PRP19, QCR2, SOD1, TCP1, TIF2, YNK1

YKL018W
CRN1, TPS3

YKL078W
NOP1, SEC27

YKL161C
GFA1, SAH1

YKL215C
HSP104

YKU70
ATP4, FRS2, HYP2, PEX19, RPT3, RVB1

YKU80
ACO1, ADR1, APT1, ARO1, ATP3, CCT3, CCT5, CLU1,

COP1, CPA2, DHH1, DPB2, ECM10, FOL2, FUN12,

GAL7, GPH1, IDH1, ILV2, LSC1, LST8, LYS12, MET16,

MKK2, MSU1, OYE2, PDX1, PHO85, PHO86, POR1,

PRE1, PST2, PUF3, PUP3, RPN12, RRP3, SIP1, SIS1,

SLC1, SLX1, SOD2, SRP54, STI1, TEM1, TFC7, TPS1,

VID31, VMA8, YBT1, YDR128W, YDR453C, YER077C,

YGR266W, YHR033W, YJR072C, YKR051W,

YLR271W, YML020W, YMR226C, YNR053C,

YOL078W, YPR003C

YLR016C
BUD13, ILS1, SMC4, SRP1

YLR074C
ADH2, COF1, CPH1, GPI15, HHF1, HMO1, HTB1,

HTB2, HYP2, LSM2, MAM33, MDH1, MGM101, OYE2,

TEF4, YBL004W, YDR036C, YFL006W, YHR052W,

YIR003W, YLR009W, YPL013C, AFG2, FYV4, HHF1,

HTA1, HTB1, HTB2, KRE32, LHP1, MAM33, NMD3,

NOG1, NOP12, NOP13, PRP43, PUF6, PWP1, RSM24,

RSM25, YBL044W, VDR036C, YDR101C, YER006W,

YGL068W, YGR103W, VHR197W, YJL122W, YPL013C

YLR097C
ADH2, CDC53, GUF1, IDH1, SKP1, UBI4

YLR186W
CAR2, OYE2, PHO81, YER030W, YPL004C

YLR222C
ARP10, DIP2, DIP5, FET4, MUM2, PGM2, POR1, SRV2,

TFP1, YHR020W, YJL069C

YLR238W
FET4, PHO84, VMR029C

YLR247C
CIN5, EXO70, HHF1, HTA1

YLR320W
ESC4, GDH2, RTT101

YLR352W
CDC53, SKP1

YLR427W
ARE1, CDC33, FET4, FUN12, GRS1, HAS1, IMD2,

IMD3, IMD4, KRE33, KRI1, MGM101, MSC3, NOP12,

NOP4, NPL3, OYE2, PDC6, TIF4631, TIF4632,

YGR090W, YHR199C, YKR081C, YOR206W, YPL012W

YML029W
PEX6, PIM1, YLR106C

YML088W
CDC53, SKP1

YMR049C
ACO1, CCT6, CDC14, EGD2, GND1, HAS1, HXT7,

MET6, MRT4, MUB1, NOG1, PRP43, SAH1, SCS2,

SEC53, SPB4, SSQ1, TIF6, UBR2, YER008W,

YGL111W, YGL245W, YLR002C, YOR206W, YTM1,

ARP2, BRX1, CRN1, EBP2, EXG1, FPR4, MRT4,

MYO1, NMD3, NOG1, NOP2, PIB2, RLP7, SCS2,

TIF6, YDR412W, YER002W, YGR103W, YHR052W,

YKR081C, YLR002C, YNL110C

YMR093W
ERB1, ROK1, YBR281C, YHR052W, YJL109C

YMR291W
FUM1, VPS33

YNL035C
KIN1

YNL056W
YNL099C

YNL094W
ABP1, COF1, CPH1, FYV8, MDH1, OYE2, PGM2,

YPL004C

YWL099C
SW14

YNL116W
PHO84, STH1, YNL311C, YPL110C

YNL157W
CPH1, HTB2, SAH1

YNL182C
APG17, HHF1, Q0032

YNL260C
POR1, YNL008C

YNL311C
ERG1, RPT2, RPT4, RVB1, SKP1, STI1, UBI4,

YHR115C, YNL116W

YOL045W
FUN30, FUN31

YOL054W
EDE1, GAC1, HHF1, HTA1, HTA2, HTB1, HTB2, KNS1,

MAM33, POB3, SPT16, YCR030C, YOR056C

YOL087C
ATP3, BEM2, COP1, EDE1, FOL2, HTB2, LHS1, NIP1,

POR1, RPG1, SES1, SRV2, TIF34, TIF35, UBI4

YOL128C
GSP2, MDH1, OYE2, TRR1

YOR026W
GSY2, Q0092

YOR227W
GLC7

YOR353C
KIC1

YPK2
CDC33, PET112, PRB1, SNF1, TFP1, YEL023C,

YGR016W

YPL150W
ARO4, CAR2, NAP1, OYE2, YGR086C, YPL004C

YPL170W
GUF1, PMA1

YPL236C
UFD2

YPR015C
CLU1, MAM33, PGM2, RET1, SXM1, YHR046C

YPR093C
FPR1, RPB11, RPB3, RPB9, RPO21, YOR131C

YPT1
DSS4, GDI1, MRS8, SEC4

YPT10
GDI1, MRS6

YPT33
BCY1, CDC33, MRS6, POR1, TPK1, TPK3, VPS21,

YNL227C, YPT52

YPT6
ACO1, GDI1, RGP1, RIC1, RNA1

YRB2
ORM1, DIA4, PRSS

YTA6
TOP2, YGR086C, YPL004C

YTMI
ERB1, RPF1, SRP54, VPS35, YBR242W, YHR052W,

YIL1370, YPD1

[0362] Bold protein names indicate those for which an interaction with the bait was confirmed in the literature using PreBIND.

3TABLE 3Comparison of HMS-PCI and HTP-Y2H datasetsDatasheetInteractions found in literatureHTP-MS/MS Spoke166HTP-MS/MS Matrix230Ito et al.46 47Uetz et al.7 51

[0363]

4

TABLE 4A

Proteins removed by filtering criteria (Protocol A)

ORF Name
Gene
Description

YLR044C
PDC1
pyruvate decarboxylase

YIL107C
PFK26
6-Phosphofructose-2-kinase

YAL005C
SSA1
Heat shock protein of HSP70 family,

cytoplasmic

YLR259C
HSP60
mitochondrial chaperonin, homolog of E.

coli
groEL protein

YJR045C
SSC1
Mitochondrial matrix protein involved in

protein import\; subunit of SceI endonuclease

YOL145C
CTR9
involved in mitosis and chromosome

segregation

YDR499W
LCD1

YMR116C
ASC1
G-beta like protein

YJR121W
ATP2
F(1)F(0)-ATPase complex beta subunit,

mitochondrial

YOL086C
ADH1
Alcohol dehydrogenase

YLL024C
SSA2
member of 70 kDa heat shock protein family

YBR196C
PGI1
Glucose-6-phosphate isomerase

YBL099W
ATP1
mitochondrial F1F0-ATPase alpha subunit

YBR118W
TEF2
translational elongation factor EF-1 alpha

YOL055C
TH120
THI for thiamine metabolism. Transcribed in

the presence of low level of thiamine (10-

8M) and turned off in the presence of high

level (10-6M) of thiamine. Under the

positive control of TH12 and TH13.

YNL064C
YDJ1
yeast dnaJ homolog (nuclear envelope

protein)\; heat shock protein

YHR111W
YHR111W
moeB, thiF, UBA1

YGL244W
RTF1
Nuclear protein

YPL106C
SSE1
HSP70 family member, highly homologous

to Ssa1p and Sse2p

YHR174W
ENO2
enolase

YCR012W
PGK1
3-phosphoglycerate kinase

YFR053C
HXK1
Hexokinase I (PI) (also called Hexokinase A)

YKL152C
GPM1
Phosphoglycerate mutase

YCL018W
LEU2
beta-IPM (isopropylmalate) dehydrogenase

YBR072W
HSP26
heat shock protein 26

YFL039C
ACT1
Actin

YBR127C
VMA2
vacuolar ATPase V1 domain subunit B

(60 kDa)

YLR180W
SAM1
S-adenosylmethionine synthetase

YBR020W
GAL1
galactokinase

YGR192C
TDH3
Glyceraldehyde-3-phosphate

dehydrogenase 3

YBR136W
MEC1
similar to phosphatidylinositol(PI)3-kinases

required for DNA damage induced

checkpoint responses in G1, S\/M, intra S,

and G2\/M in mitosis

YFL037W
TUB2
beta-tubulin

YJL008C
CCT8
Component of Chaperonin Containing

T-complex subunit eight

YGL009C
LEU1
isopropylmalate isomerase

YDR050C
TPI1
triosephosphate isomerase

YDL126C
CDC48
microsomal ATPase

YLR150W
STM1
gene product has affinity for quadruplex

nucleic acids

YAL038W
CDC19
Pyruvate kinase

YML085C
TUB1
alpha-tubulin

YJL148W
RPA34
unshared RNA polymerase I subunit

YBR221C
PDB1
beta subunit of pyruvate dehydrogenase

(E1 beta)

YJL088W
ARG3
Ornithine carbamoyltransferase

YMR186W
HSC82
constitutively expressed heat shock protein

YBR035C
PDX3
pyridoxine (pyridoxiamine) phosphate

oxidase

YLR418C
CDC73
RNA polymerase II accessory protein

YJL130C
URA2
carbamoyl-phophate synthetase, aspartate

transcarbamylase, and glutamine

amidotransferase

YER177W
BMH1
Homolog of mammalian 14-3-3 proteins

YMR205C
PFK2
phosphofructokinase beta subunit

YCL040W
GLK1
Glucokinase

YDL055C
PSA1
mannose-1-phosphate guanyltransferase,

GDP-mannose pyrophosphorylase

YLR340W
RPP0
60S ribosomal protein P0 (A0) (L10E)

YKL060C
FBA1
aldolase

YGR254W
ENO1
enolase I

YJR123W
RPS5
Ribosomal protein S5 (S2) (rp14) (YS8)

YBR279W
PAF1
RNA polymerase II-associated protein

YDL229W
SSB1
cytoplasmic member of the HSP70 family

YER165W
PAB1
Poly(A) binding protein, cytoplasmic and

nuclear

YNL178W
RPS3
Ribosomal protein S3 (rp13) (YS3)

YBR181C
RPS6B
40S ribosomal gene product S6B (S10B)

(rp9) (YS4)

YGL206C
CHC1
presumed vesicle coat protein

YPL061W
ALD6
Cytosolic Aldehyde Dehydrogenase

YGL173C
KEM1
cytoplsamic 5′-to-3′ exonuclease.

YFL018c
LPD1
dihydrolipoamide dehydrogenase precursor

(mature protein is the E3 component of

alpha-ketoacid dehydrogenase complexes)

YNL071W
LAT1
Dihydrolipoamide acetyltransferase

component (E2) of pyruvate dehydrogenase

complex

YPL235W
RVB2
RUVB-like protein

YGL253W
HXK2
Hexokinase II (PII) (also called

Hexokinase B)

YPL258C
TH121
THI for thiamine metabolism. Transcribed in

the presence of low level of thiamine (10-

8M) and turned off in the presence of high

level (10-6M) of thiamine. Under the

positive control of THI2 and THI3.

YPL240C
HSP82
82 kDa heat shock protein\; homolog of

mammalian Hsp90

YOR063W
RPL3
Ribosomal protein L3 (rp1) (YL1)

YPL131W
RPL5
Ribosomal protein L5 (L1a) (YL3)

YJR009C
TDH2
glyceraldehyde 3-phosphate dehydrogenase

YHR082C
KSP1
Ser\/Thr protein kinase

YNL209W
SSB2
Heat shock protein of HSP70 family,

homolog of SSB1

YMR076C
PDS5
(putative) involved in sister chromosome

cohesion during mitosis

YBR031W
RPL4A
Ribosomal protein L4A (L2A) (rp2) (YL2)

YJL034W
KAR2
Homologue of mammalian BiP (GPR78)

protein\; member of the HSP70 gene family

YDR385W
EFT2
translation elongation factor 2 (EF-2)

YDR171W
HSP42
heat shock protein similar to HSP26,

involved in cytoskeleton assembly

YJR077C
MIR1

YHR203C
RPS4B
Ribosomal protein S4B (YS6) (rp5) (S7B)

YFR031C-A
RPL2A
Ribosomal protein L2A (L5A) (rp8) (YL6)

YJL066C
YJL066C

YLL045C
RPL8B
Ribosomal protein L8B (L4B) (rp6) (YL5)

YHL034C
SBP1
Single-strand nucleic acid binding protein

YDR099W
BMH2
member of conserved eukaryotic 14-3-3 gene

family

YML028W
TSA1
thioredoxin-peroxidase (TPx)\; reduces

H2O2 and alkyl hydroperoxides with the use

of hydrogens provided by thioredoxin,

thioredoxin reductase, and NADPH

YBL072C
RPS8A
Ribosomal protein S8A (S14A) (rp19) (YS9)

YLR249W
YEF3
EF-3 (translational elongation factor 3)

YDR502C
SAM2
S-adenosylmethionine synthetase

YMR214W
SCJ1
dnaJ homolog

YER110C
KAP123
Karyopherin beta 4

YOR151C
RPB2
second largest subunit of RNA polymerase II

YGL048C
RPT6
ATPase

YJL052W
TDH1
Glyceraldehyde-3-phosphate

dehydrogenase I

YKL180W
RPL17A
Ribosomal protein L17A (L20A) (YL17)

YML124C
TUB3
alpha-tubulin

YGL076C
RPL7A
Ribosomal protein L7A (L6A) (rp11) (YL8)

YFL016C
MDJ1
DnaJ homolog involved in mitochondrial

biogenesis and protein folding

YCL064C
CHA1
catabolic serine (threonine) dehydratase

YMR066W
SOV1
(putative) involved in respiration

YDR148C
KGD2
dihydrolipoyl transsuccinylase component of

alpha-ketoglutarate dehydrogenase complex

in mitochondria

YKL035W
UGP1
Uridinephosphoglucose pyrophosphorylase

YOR374W
ALD4
mitochondrial aldehyde dehydrogenase

YKL182W
FAS1
pentafunctional enzyme consisting of the

following domains: acetyl transferase, enoyl

reductase, dehydratase and malonyl\/palmityl

transferase

YCL037C
SRO9
RNA binding protein with La motif

YBL030C
PET9
mitochondrial ADP\/ATP translocator

YHL033C
RPL8A
Ribosomal protein L8A (rp6) (YL5) (L4A)

YIL075C
RPN2
RPN2p is a component of the 26S proteosome

YGL123W
RPS2
Ribosomal protein S2 (S4) (rp12) (YS5)

YBR019C
GAL10
UDP-glucose 4-epimerase

YJL177W
RPL17B
Ribosomal protein L17B (L20B) (YL17)

YPL231W
FAS2
alpha subunit of fatty acid synthase

YGR282C
BGL2
Cell wall endo-beta-1,3-glucanase

YER178W
PDA1
alpha subunit of pyruvate dehydrogenase

(E1 alpha)

YNR001C
CIT1
citrate synthase. Nuclear encoded

mitochondrial protein.

YJL111W
CCT7
Component of Chaperonin Containing

T-complex subunit seven

YDL143W
CCT4
component of chaperonin complex

YGL135W
RPL1B
Ribosomal protein L1B

[0364]

5

TABLE 4B

Proteins removed by filtering criteria (Protocol B).

ORF
Gene Name
Description

YGL009C
LEU1
isopropylmalate isomerase

YAL005C
SSA1
Heat shock protein of HSP70 family,

cytoplasmic

YOL055C
TH120
THI for thiamine metabolism. Transcribed in

the presence of low level of thiamine (10-

8M) and turned off in the presence of high

level (10-6M) of thiamine. Under the

positive control of THI2 and THI3.

YCL018W
LEU2
beta-IPM (isopropylmalate) dehydrogenase

YLL024C
SSA2
member of 70 kDa heat shock protein family

YAL038W
CDC19
Pyruvate kinase

YLR044C
PDC1
pyruvate decarboxylase

YHR174W
ENO2
enolase

YGR192C
TDH3
Glyceraldehyde-3-phosphate dehydrogenase 3

YGR254W
ENO1
enolase I

YBR118W
TEF2
translational elongation factor EF-1 alpha

YOL086C
ADH1
Alcohol dehydrogenase

YGL244W
RTF1
Nuclear protein

YCR012W
PGK1
3-phosphoglycerate kinase

YLR259C
HSP60
mitochondrial chaperonin, homolog of E.

coli
groEL protein

YPL106C
SSE1
HSP70 family member, highly homologous

to Ssa1p and Sse2p

YMR116C
ASC1
G-beta like protein

YDL229W
SSB1
cytoplasmic member of the HSP70 family

YJL052W
TDH1
Glyceraldehyde-3-phosphate

dehydrogenase 1

YJR045C
SSC1
Mitochondrial matrix protein involved in

protein import\; subunit of SceI

endonuclease

YKL060C
FBA1
aldolase

YKL152C
GPM1
Phosphoglycerate mutase

YBR072W
HSP26
heat shock protein 26

YMR186W
HSC82
constitutively expressed heat shock protein

YER091C
MET6
vitamin B12-(cobalamin)-independent

isozyme of methionine synthase (also called

N5-methyltetrahydrofolate homocysteine

methyltransferase or 5-methyltetra-

hydropteroyl triglutamate homocysteine

methyltransferase)

YBL075C
SSA3
heat-inducible cytosolic member of the 70

kDa heat shock protein family

YBR196C
PGII
Glucose-6-phosphate isomerase

YDR502C
SAM2
S-adenosylmethionine synthetase

YDR099W
BMH2
member of conserved eukaryotic 14-3-3 gene

family

YLR340W
RPP0
60S ribosomal protein P0 (A0) (L10E)

YGR214W
RPS0A
Ribosomal protein S0A

YLR180W
SAM1
S-adenosylmethionine synthetase

YBRC19C
GAL10
UDP-glucose 4-epimerase

YNL209W
SSB2
Heat shock protein of HSP70 family,

homolog of SSB1

YJR121W
ATP2
F(1)F(0)-ATPase complex beta subunit,

mitochondrial

YOR308C
SNU66
66 kD U4\/U6.U5 snRNP associated protein

YJR009C
TDH2
glyceraldehyde 3-phosphate dehydrogenase

YJL034W
KAR2
Homologue of mammalian BiP (GPR78)

protein\; member of the HSP70 gene family

YMR108W
ILV2
acetolactate synthase

YER177W
BMHI
Homolog of mammalian 14-3-3 proteins

YDR050C
TPI1
triosephosphate isomerase

YBR127C
VMA2
vacuolar ATPase V1 domain subunit B

(60 kDa)

YGR171C
MSM1
mitochondrial methionyl-tRNA synthetase

YNL178W
RPS3
Ribosomal protein S3 (rp13) (YS3)

YER043C
SAH1
putative S-adenosyl-L-homocysteine

hydrolase

YLR355C
ILV5
acetohydroxyacid reductoisomerase

YDR171W
HSP42
heat shock protein similar to HSP26,

involved in cytoskeleton assembly

YHR020W
YHR020W
Aminoacyl tRNA-synthetase

YBR020W
GAL1
galactokinase

YMR319C
FET4
Low-affinity Fe(II) transport protein

YDL055C
PSA1
mannose-1-phosphate guanyltransferase,

GDP-mannose pyrophosphorylase

YKL182W
FAS1
pentafunctional enzyme consisting of the

following domains: acetyl transferase, enoyl

reductase, dehydratase and malonyl\/palmityl

transferase

YJR123W
RPS5
Ribosomal protein S5 (S2) (rp14) (YS8)

YPL231W
FAS2
alpha subunit of fatty acid synthase

YHR111W
YHR111W
moeB, thiF, UBA1

YFR053C
HXK1
Hexokinase I (PI) (also called Hexokinase A)

YKL104C
GFA1
Glutamine_fructose-6-phosphate

amidotransferase (glucoseamine-6-phosphate

synthase)

YBL099W
ATP1
mitochondrial F1F0-ATPase alpha subunit

YPL131W
RPL5
Ribosomal protein L5 (L1a)(YL3)

YLR249W
YEF3
EF-3 (translational elongation factor 3)

YER103W
SSA4
member of 70 kDa heat shock protein family

YBR031W
RPL4A
Ribosomal protein L4A (L2A) (rp2) (YL2)

YOR375C
GDH1
NADP-specific glutamate dehydrogenase

YDL126C
CDC48
microsomal ATPase

YGL206C
CHC1
presumed vesicle coat protein

YOR374W
ALD4
mitochondrial aldehyde dehydrogenase

YFL039C
ACT1
Actin

YHR203C
RPS4B
Ribosomal protein S4B (YS6) (rp5) (S7B)

YFR031C-A
RPL2A
Ribosomal protein L2A (L5A) (rp8) (YL6)

YLR048W
RPS0B
Ribosomal protein S0B

YGL048C
RPT6
ATPase

YJL130C
URA2
carbamoyl-phophate synthetase, aspartate

transcarbamylase, and glutamine

amidotransferase

YBR181C
RPS6B
40S ribosomal gene product S6B (S10B)

(rp9) (YS4)

YDR394W
RPT3
ATPase (AAA family) component of the 26S

proteasome complex

YJL008C
CCT8
Component of Chaperonin Containing

T-complex subunit eight

YJL153C
INO1
L-myo-inositol-1-phosphate synthase

YJL117W
PHO86
Putative inorganic phosphate transporter

YOL145C
CTR9
involved in mitosis and chromosome

segregation

YPL137C
YPL137C

YGR240C
PFK1
phosphofructokinase alpha subunit

YGL245W
YGL245W

YBL039C
URA7
CTP synthase, highly homologus to URA8

CTP synthase

YGL008C
PMA1
plasma membrane H+-ATPase

YER110C
KAP123
Karyopherin beta 4

YGL253W
HXK2
Hexokinase II (PII) (also called

Hexokinase B)

YHR199C
YHR199C

YDR385W
EFT2
translation elongation factor 2 (EF-2)

YJL138C
TIF2
translation initiation factor eIF4A

YPL061W
ALD6
Cytosolic Aldehyde Dehydrogenase

YHR137W
ARO9
aromatic amino acid aminotransferase II

YML028W
TSA1
thioredoxin-peroxidase (TPx)\; reduces

H2O2 and alkyl hydroperoxides with the use

of hydrogens provided by thioredoxin,

thioredoxin reductase, and NADPH

YMR257C
PET111
translational activator of cytochrome c

oxidase subunit II

YOR136W
IDH2
NAD+-dependent isocitrate dehydrogenase

YOR117W
RPT5
26S protease regulatory subunit

YFL037W
TUB2
beta-tubulin

YOR063W
RPL3
Ribosomal protein L3 (rp1) (YL1)

YNL037C
IDH1
alpha-4-beta-4 subunit of mitochondrial

isocitrate dehydrogenase 1

YCL043C
PDI1
protein disulfide isomerase

YML063W
RPS1B
Ribosomal protein S1B (rp10B)

YBR018C
GAL7
galactose-1-phosphate uridyl transferase

YJR077C
MIR1

YCL061C
MRC1

YLR134W
PDC5
pyruvate decarboxylase

YHL033C
RPL8A
Ribosomal protein L8A (rp6) (YL5) (L4A)

YDR012W
RPL4B
Ribosomal protein L4B (L2B) (rp2) (YL2)

YPL240C
HSP82
82 kDa heat shock protein\; homolog of

mammalian Hsp90

YBL072C
RPS8A
Ribosomal protein S8A (S14A) (rp19) (YS9)

YDL083C
RPS16B
Ribosomal protein S16B (rp61R)

YJR109C
CPA2
carbamyl phosphate synthetase

YGL076C
RPL7A
Ribosomal protein L7A (L6A) (rp11) (YL8)

YLR304C
ACO1
Aconitase, mitochondrial

YDL143W
CCT4
component of chaperonin complex

YDL185W
TFP1
vacuolar ATPase V1 domain subunit A

(69 kDa)

YOR123C
LEO1

YOR096W
RPS7A
Ribosomal protein S7A (rp30)

YGR094W
VAS1
mitochondrial and cytoplasmic valyl-tRNA

synthetase

YBR169C
SSE2
HSP70 family member, highly homologous

to Sse1p

YBR011C
IPP1
Inorganic pyrophosphatase

YDR018C
YDR018C

YKL035W
UGP1
Uridinephosphoglucose pyrophosphorylase

YFR030W
MET10
subunit of assimilatory sulfite reductase

YKL081W
TEF4
Translation elongation factor EF-1gamma

YJR104C
SOD1
Cu, Zn superoxide dismutase

YHL015W
RPS20
Ribosomal protein S20

YPL258C
THI21
THI for thiamine metabolism. Transcribed in

the presence of low level of thiamine

(10-8M) and turned off in the presence of

high level (10-6M) of thiamine. Under the

positive control of THI2 and THI3.

YHR027C
RPN1
Subunit of 26S Proteasome (PA700 subunit)

YNL055C
POR1
Outer mitochondrial membrane porin

(voltage-dependent anion channel, or

VDAC)

YLR441C
RPS1A
Ribosomal protein S1A (rp10A)

YLR354C
TAL1
Transaldolase, enzyme in the pentose

phosphate pathway

YGL062W
PYC1
pyruvate carboxylase

YDR190C
RVB1
RUVB-like protein

YCL040W
GLK1
Glucokinase

YBL021C
HAP3
transcriptional activator protein of CYC1

YBR218C
PYC2
pyruvate carboxylase

YLR058C
SHM2
serine hydroxymethyltransferase

YDR477W
SNF1
protein serine\/threonine kinase

YGR085C
RPL11B
60S ribosomal protein L11B (L16B)

(rp39B) (YL22)

YDR158W
HOM2
aspartic beta semi-aldehyde dehydrogenase

YPR159W
KRE6
potential beta-glucan synthase

YIL107C
PFK26
6-Phosphofructose-2-kinase

YGL234W
ADE5, 7
glycinamide ribotide synthetase and

aminoimidazole ribotide synthetase

YOR369C
RPS12
40S ribosomal protein S12

YMR247C
YMR247C

YBR189W
RPS9B
Ribosomal protein S9B (S13) (rp21) (YS11)

YLL026W
HSP104
104 kDa heat shock protein

YDL007W
RPT2
(putative) 26S protease subunit

YOR261C
RPN8
Subunit of the regulatory particle of the

proteasome

YIL142W
CCT2
molecular chaperone

YFL045C
SEC53
phosphomannomutase

YNL064C
YDJ1
yeast dnaJ homolog (nuclear envelope

protein)\; heat shock protein

YKL022C
CDC16
putative metal-binding nucleic acid-binding

protein, interacts with Cdc23p and Cdc27p to

catalyze the conjugation of ubiquitin to

cyclin B

YNL040W
YNL040W

YML085C
TUB1
alpha-tubulin

YIL033C
BCY1
regulatory subunit of cAMP-dependent

protein kinase

YGR180C
RNR4
Ribonucleotide Reductase

YDR064W
RPS13
Ribosomal protein S13 (S27a) (YS15)

YCR002C
CDC10
conserved potential GTP-ginding protein

YML124C
TUB3
alpha-tubulin

YIL094C
LYS12
Homo-isocitrate dehydrogenase

YLR153C
ACS2
acetyl-coenzyme A synthetase

YPR074C
TKL1
Transketolase 1

YDR212W
TCP1
chaperonin subunit alpha

YDR155C
CPH1
cyclophilin peptidyl-prolyl cis-trans

isomerase

YHR183W
GND1
Phosphogluconate Dehydrogenase

(Decarboxylating)

YJR139C
HOM6
Homoserine dehydrogenase

(L-homoserine:NADP oxidoreductase)

YER062C
HOR2
DL-glycerol-3-phosphatase

YJR064W
CCT5
subunit of chaperonin subunit epsilon

YDR447C
RPS17B
Ribosomal protein S17B (rp51B)

YGL026C
TRP5
tryptophan synthetase

YOL139C
CDC33
mRNA cap binding protein eIF-4E

YDR450W
RPS18A
Ribosomal protein S18A

YER074W
RPS24A
40S ribosomal protein S24A

YNR001C
CIT1
citrate synthase. Nuclear encoded

mitochondrial protein.

YDL082W
RPL13A
Ribosomal protein LI3A

YLR150W
STM1
gene product has affinity for quadruplex

nucleic acids

YGL147C
RPL9A
Ribosomal protein L9A (L8A) (rp24) (YL11)

YBR025C
YBR025C
probable purine nucleotide-binding protein

YGL135W
RPL1B
Ribosomal protein L1B

YGL105W
ARC1
G4 nucleic acid binding protein, involved in

tRNA aminoacylation

YHR179W
OYE2
NAPDH dehydrogenase (old yellow

enzyme), isoform 2

YDL182W
LYS20
homocitrate synthase, highly homologous

to YDL131W

YJL066C
MPM1

YBR279W
PAF1
RNA polymerase II-associated protein

YIL053W
RHR2
DL-glycerol-3-phosphatase

YEL051W
VMA8
vacuolar ATPase V1 domain subunit D

YMR205C
PFK2
phosphofructokinase beta subunit

YMR120C
ADE17
5-aminoimidazole-4-carboxamide

ribonucleotide (AICAR)

transformylase\/IMP cyclohydrolase

YDR148C
KGD2
dihydrolipoyl tranasuccinylase component

of alpha-ketoglutarate dehydrogenase

complex in mitochondria

YMR145C
YMR145C

YNR058W
BIO3
7,8-diamino-pelargonic acid

aminotransferase (DAPA) aminotransferase

YCR084C
TUP1
glucose repression regulatory protein,

exhibits similarity to beta subunits of G

proteins

YLL045C
RPL8B
Ribosomal protein L8B (L4B) (rp6) (YL5)

YLL018C
DPS1
Aspartyl-tRNA synthetase, cytosolic

YGL202W
ARO8
aromatic amino acid aminotransferase

YBL076C
ILS1
cytoplasmic isoleucyl-tRNA synthetase

YLR109W
AHP1
alkyl hydroperoxide reductase

YDR279W
YDR279W

YPL110C
YPL110C

YKL210W
UBA1
ubiquitin activating enzyme, similar to

Uba2p

YPL235W
RVB2
RUVB-like protein

YMR226C
YMR226C

YBR126C
TPS1
56 kD synthase subunit of trehalose-6-

phosphate synthase\/phosphatase complex

YLR075W
RPL10
Ribosomal protein L10\; Ubiquinol-

cytochrome C reductase complex subunit VI

requiring protein

YGR155W
CYS4
Cystathionine beta-synthase

YDR427W
RPN9
Subunit of the regulatory particle of the

proteasome

YOR317W
FAA1
long chain fatty acyl:CoA synthetase

YJR105W
ADO1
adenosine kinase

YLR438W
CAR2
ornithine aminotransferase

YBR121C
GRS1
Glycyl-tRNA synthase

YLR222C
YLR222C

YMR315W
YMR315W

YER021W
RPN3
component of the regulatory module of the

26S proteasome, homologous to human p58

subunit

YGL256W
ADH4
alcohol dehydrogenase isoenzyme IV

YMR105C
PGM2
Phosphoglucomutase

YMR062C
ECM40
acetylornithine acetyltransferase

YPL028W
ERG10
acetoacetyl CoA thiolase

YER178W
PDA1
alpha subunit of pyruvate dehydrogenase

(E1 alpha)

YCR031C
RPS14A
Ribosomal protein SL4A (rp59A)

YOR259C
RPT4
ATPase\; component of the 26S proteasome

cap subunit

YJL167W
ERG20
Farnesyl diphosphate synthetase

(FPP synthetase)

YDL124W
YDL124W

YAR010C
YAR010C
TY1B

YDL225W
SHS1
Septin homolog

YFR004W
RPN11
Similar to S. pombe PAD1 gene product

YOR151C
RPB2
second largest subunit of RNA polymerase II

YOL058W
ARG1
arginosuccinate synthetase

YNL302C
RPS19B
Ribosomal protein S19B (rp55B) (S16aB)

(YS16B)

YBR048W
RPS11B
Ribosomal protein S11B (S18B) (rp41B)

(YS12)

YNL069C
RPL16B
Ribosomal protein LL6B (L2LB) (rp23)

(YL15)

YPR191W
QCR2
40 kDa ubiquinol cytochrome-c reductase

core protein 2

YDR471W
RPL27B
Ribosomal protein L27B

YCR053W
THR4
threonine synthase

YGL123W
RPS2
Ribosomal protein S2 (S4) (rp12)

(YS5)

YJL026W
RNR2
small subunit of ribonucleotide reductase

YOL138C
YOL138C

YJR070C
YJR070C

YBL027W
RPL19B
Ribosomal protein L19B (YL14) (L23B)

(rp15L)

YBR221C
PDB1
beta subunit of pyruvate dehydrogenase

(E1 beta)

YDR127W
ARO1
pentafunctional arom polypeptide (contains:

3-dehydroquinate synthase, 3-dehydroquinate

dehydratase (3-dehydroquinase), shikimate

5-dehydrogenase, shikimate kinase, and

epap synthase)

YDL097C
RPN6
Subunit of the regulatory particle of the

proteasome

YEL060C
PRB1
vacuolar protease B

YDR418W
RPL12B
Ribosomal protein LL2B (L15B) (YL23)

YLR448W
RPL6B
60S ribosomal subunit protein L6B (L17B)

(rp18) (YL16)

YDR129C
SAC6
fibrim homolog (actin-filament bundling

protein)

YHR082C
KSP1
Ser\/Thr protein kinase

YDR342C
HXT7
Hexose transporter

[0365]

6

TABLE 5A

Proteins identified in control lanes (Protocol A).

ORF Name
Gene
Description

YOL086C
ADH1
Alcohol dehydrogenase

YMR116C
ASC1
G-beta like protein

YAL038W
CDC19
Pyruvate kinase

YLR418C
CDC73
RNA polymerase II accessory protein

YOL145C
CTR9
involved in mitosis and chromosome

segregation

YDR385W
EFT2
translation elongation factor 2 (EF-2)

YGR254W
ENO1
enolase I

YHR174W
ENO2
enolase

YPL231W
FAS2
alpha subunit of fatty acid synthase

YKL060C
FBA1
aldolase

YMR186W
HSC82
constitutively expressed heat shock

protein

YGL253W
HXK2
Hexokinase II (PII) (also called Hexokinase B)

YDR499W
LCD1

YOR123C
LEO1

YGL009C
LEU1
isopropylmalate isomerase

YBR136W
MEC1
similar to phosphatidylinositol(PI)3-

kinases required for DNA damage

induced checkpoint responses in G1,

S\/M, intra S, and G2\/M in mitosis

YBR279W
PAF1
RNA polymerase II-associated protein

YBR221C
PDB1
beta subunit of pyruvate dehydrogenase

(E1 beta)

YLR044C
PDC1
pyruvate decarboxylase

YMR076C
PDS5
(putative) involved in sister

chromosome cohesion during mitosis

YBR035C
PDX3
pyridoxine (pyridoxiamine) phosphate

oxidase

YIL107C
PFK26
6-Phosphofructose-2-kinase

YCR012W
PGK1
3-phosphoglycerate kinase

YAR007C
RFA1
69 kDa subunit of the heterotrimeric

RPA (RF-A) single-stranded DNA

binding protein, binds URS1 and

CAR1

YNL312W
RFA2
subunit 2 of replication factor RF-A\;

29\% identical to the human p34 subunit

of RF-A

YJL173C
RFA3
subunit 3 of replication factor-A

YCR028C-A
RIM1
Single-stranded zinc finger DNA-

binding protein

YGR180C
RNR4
Ribonucleotide Reductase

YJL148W
RPA34
unshared RNA polymerase I subunit

YPR102C
RPL11A
Ribosomal protein L11A (L16A)

(rp39A) (YL22)

YGR085C
RPL11B
60S ribosomal protein L11B (L16B)

(rp39B) (YL22)

YDR418W
RPL12B
Ribosomal protein L12B (L15B)

(YL23)

YDL082W
RPL13A
Ribosomal protein L13A

YNL069C
RPL16B
Ribosomal protein L16B (L21B)

(rp23) (YL15)

YKL180W
RPL17A
Ribosomal protein L17A (L20A)

(YL17)

YJL177W
RPL17B
Ribosomal protein L17B (L20B)

(YL17)

YBL027W
RPL19B
Ribosomal protein L19B (YL14)

(L23B) (rp15L)

YGL135W
RPL1B
Ribosomal protein L1B

YMR242C
RPL20A
Ribosomal protein L20A (L18A)

YOR312C
RPL20B
60S ribosomal protein L20B (L18B)

YBR191W
RPL21A
Ribosomal protein L21A

YPL079W
RPL21B
Ribosomal protein L21B

YBL087C
RPL23A
Ribosomal protein L23A (L17aA)

(YL32)

YOL127W
RPL25
Ribosomal protein L25 (rp16L)

(YL25)

YHR010W
RPL27A
Ribosomal protein L27A

YDR471W
RPL27B
Ribosomal protein L27B

YIL018W
RPL2B
Ribosomal protein L2B (L5B) (rp8)

(YL6)

YOR063W
RPL3
Ribosomal protein L3 (rp1) (YL1)

YGL030W
RPL30
Large ribosomal subunit protein L30

(L32) (rp73) (YL38)

YPL143W
RPL33A
Ribosomal protein L33A (L37A)

(YL37) (rp47)

YDL191W
RPL35A
Ribosomal protein L35A

YJR094W-A
RPL43B
Ribosomal protein L43B

YBR031W
RPL4A
Ribosomal protein L4A (L2A) (rp2)

(YL2)

YDR012W
RPL4B
Ribosomal protein LAB (L2B) (rp2)

(YL2)

YPL131W
RPL5
Ribosomal protein L5 (L1a) (YL3)

YML073C
RPL6A
Ribosomal protein L6A (L17A) (rp18)

(YL16)

YLR448W
RPL6B
60S ribosomal subunit protein L6B

(L17B) (rp18) (YL16)

YGL076C
RPL7A
Ribosomal protein L7A (L6A) (rp11)

(YL8)

YPL198W
RPL7B
Ribosomal protein L7B (L6B) (rp11)

(YL8)

YHL033C
RPL8A
Ribosomal protein L8A (rp6) (YL5)

(L4A)

YGL147C
RPL9A
Ribosomal protein L9A (L8A) (rp24)

(YL11)

YLR340W
RPP0
60S ribosomal protein P0 (A0) (L10E)

YGR214W
RPS0A
Ribosomal protein S0A

YLR048W
RPS0B
Ribosomal protein S0B

YOR293W
RPS10A
Ribosomal protein S10A

YBR048W
RPS11B
Ribosomal protein S11B (S18B)

(rp41B) (YS12)

YOR369C
RPSL2
40S ribosomal protein S12

YDR064W
RPS13
Ribosomal protein S13 (S27a) (YS15)

YOL040C
RPS15
40S ribosomal protein S15 (S21) (rp52)

(RIG protein)

YDL083C
RPS16B
Ribosomal protein S16B (rp61R)

YML024W
RPS17A
Ribosomal protein S17A (rp51A)

YDR447C
RPS17B
Ribosomal protein S17B (rp51B)

YDR450W
RPS18A
Ribosomal protein S18A

YLR441C
RPS1A
Ribosomal protein S1A (rp10A)

YML063W
RPS1B
Ribosomal protein S1B (rp10B)

YGL123W
RPS2
Ribosomal protein S2 (S4) (rp12) (YS5)

YHL015W
RPS20
Ribosomal protein S20

YJL190C
RPS22A
Ribosomal protein S22A (S24A) (rp50)

(YS22)

YER074W
RPS24A
40S ribosomal protein S24A

YGR027C
RPS25A
Ribosomal protein S25A (S31A) (rp45)

(YS23)

YNL178W
RPS3
Ribosomal protein S3 (rp13) (YS3)

YHR203C
RPS4B
Ribosomal protein S4B (YS6) (rp5)

(S7B)

YJR123W
RPS5
Ribosomal protein S5 (S2) (rp14)

(YS8)

YPL090C
RPS6A
Ribosomal protein S6A (S10A) (rp9)

(YS4)

YBR181C
RPS6B
40S ribosomal gene product S6B

(S10B) (rp9) (YS4)

YBL072C
RPS8A
Ribosomal protein S8A (S14A) (rp19)

(YS9)

YPL081W
RPS9A
Ribosomal protein S9A (S13) (rp21)

(YS11)

YGL244W
RTF1
Nuclear protein

YAL005C
SSA1
Heat shock protein of HSP70 family,

cytoplasmic

YLL024C
SSA2
member of 70 kDa heat shock protein

family

YBL075C
SSA3
heat-inducible cytosolic member of the

70 kDa heat shock protein family

YER103W
SSA4
member of 70 kDa heat shock protein

family

YDL229W
SSB1
cytoplasmic member of the HSP70

family

YNL209W
SSB2
Heat shock protein of HSP70 family,

homolog of SSB1

YPL106C
SSE1
HSP70 family member, highly

homologous to Ssa1p and Sse2p

YBR169C
SSE2
HSP70 family member, highly

homologous to Sse1p

YLR150W
STM1
gene product has affinity for quadruplex

nucleic acids

YJL052W
TDH1
Glyceraldehyde-3-phosphate

dehydrogenase 1

YJR009C
TDH2
glyceraldehyde 3-phosphate

dehydrogenase

YGR192C
TDH3
Glyceraldehyde-3-phosphate

dehydrogenase 3

YDR050C
TPI1
triosephosphate isomerase

YML028W
TSA1
thioredoxin-peroxidase (TPx)\;

reduces H2O2 and alkyl hydroperoxides

with the use of hydrogens provided by

thioredoxin, thioredoxin reductase,

and NADPH

YBR012W-B
YBR012W-B
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YDR210W-D
YDR210W-D
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YDR261C-C
YDR261C-C
TyA gag protein. Gag processing

produces capsid proteins.

YDR261C-D
YDR261C-D
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YDR316W-B
YDR316W-B
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YDR365W-B
YDR365W-B
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YLR249W
YEF3
EF-3 (translational elongation factor 3)

YGR027W-B
YGR027W-B
The TyB Gag-Pol protein. Gag

processing produces capsid proteins.

Pol is cleaved to produce protease,

reverse transcriptase, and integrase

activities.

YHR111W
YHR111W
moeB, thiF, UBA1

YJR029W
YJR029W

YMR247C
YMR247C

YNL054W-A
YNL054W-A
TyA Gag protein. Gag processing

produces capsid proteins.

YOR142W-B
YOR142W-B
TyB Gag-Pol protein. Gag processing

produces capsid proteins. Pol is cleaved

to produce protease, reverse

transcriptase and integrase activities.

YPL137C
YPL137C

YPL257W-B
YPL257W-B
TyB Gag-Pol protein. Gag processing

produces capsid proteins. Pol is cleaved

to produce protease, reverse transcriptase

and integrase activities.

[0366]

7

TABLE 5B

Proteins identified in control lanes (Protocol B)

ORF Name
Gene
Description

YOL086C
ADH1
Alcohol dehydrogenase

YMR116C
ASC1
G-beta like protein

YBL099W
ATP1
mitochondrial F1F0-ATPase alpha subunit

YJR121W
ATP2
F(1)F(0)-ATPase complex beta subunit,

mitochondrial

YIL142W
CCT2
molecular chaperone

YJL014W
CCT3
Cytoplasmic chaperonin subunit gamma

YDL143W
CCT4
component of chaperonin complex

YJR064W
CCT5
subunit of chaperonin subunit epsilon

YJL111W
CCT7
Component of Chaperonin Containing

T-complex subunit seven

YJL008C
CCT8
Component of Chaperonin Containing

T-complex subunit eight

YKL022C
CDC16
putative metal-binding nucleic

acid-binding protein,

interacts with Cdc23p and Cdc27p to

catalyze the conjugation of ubiquitin

to cyclin B

YAL038W
CDCL9
Pyruvate kinase

YLR418C
CDC73
RNA polymerase II accessory protein

YGR218W
CRM1
omosome region maintenance protein

YOL145C
CTR9
involved in mitosis and chromosome

segregation

YHR174W
ENO2
enolase

YK1182W
FAS1
pentafunctional enzyme consisting of

the following domains: acetyl transferase,

enoyl reductase, dehydratase and

malonyl\/palmityl transferase

YPL231W
FAS2
alpha subunit of fatty acid synthase

YMR319C
FET4
Low-affinity Fe(II) transport protein

YGR267C
FOL2
GTP-cyclohydrolase I

YKL152C
GPM1
Phosphoglycerate mutase

YER110C
KAPL23
Karyopherinbeta4

YPR159W
KRE6
potential beta-glucan synthase

YHR082C
KSP1
Ser\/Thr protein kinase

YNL071W
LAT1
Dihydrolipoamide acetyltransferase

component (E2) of pyruvate dehydrogenase

complex

YDR499W
LCD1

YORL23C
LEO1

YGL009C
LEU1
isopropylmalate isomerase

YFR001W
LOC1
Double-stranded RNA-binding protein

YBR136W
MEC1
similar to phosphatidylinositol(PI)3-kinases

required for DNA damage induced

checkpoint responses in G1, S\/M, intra S,

and G2\/M in mitosis

YDL167C
NRP1
Asparagine-rich protein

YDR356W
NUF1
component of the spindle pole body that

interacts with Spc42p, calmodulin, and a 35

kDa protein

YBR279W
PAF1
RNA polymerase II-associated protein

YBR221C
PDB1
beta subunit of pyruvate dehydrogenase

(E1 beta)

YMR076C
PDS5
(putative) involved in sister chromosome

cohesion during mitosis

YIL107C
PFK26
6-Phosphofructose-2-kinase

YCR012W
PGK1
3-phosphoglycerate kinase

YNL055C
POR1
Outer mitochondrial membrane porin

(voltage-dependent anion channel, or

VDAC)

YDL055C
PSA1
mannose-1-phosphate guanyltransferase,

GDP-mannose pyrophosphorylase

Q0255
Q0255

YJL173C
RFA3
subunit 3 of replication factor-A

YCR028C-A
RIM1
Single-stranded zinc finger DNA-binding

protein

YJL148W
RPA34
unshared RNA polymerase I subunit

YOR151C
RPB2
second largest subunit of RNA polymerase II

YLR075W
RPL10
Ribosomal protein L10\; Ubiquinol-

cytochrome C reductase complex subunit VI

requiring protein

YGR085C
RPL11B
60S ribosomal protein L11B (L16B) (rp39B)

(YL22)

YDR418W
RPL12B
Ribosomal protein L12B (L15B) (YL23)

YMR142C
RPL13B
Ribosomal protein L13B

YHL001W
RPL14B
Ribosomal protein L14B

YLR029C
RPL15A
Ribosomal protein L15A (YL10) (rp15R)

(L13A)

YIL133C
RPL16A
Ribosomal protein L16A (L21A) (rp22)

(YL15)

YNL069C
RPL16B
Ribosomal protein L16B (L21B) (rp23)

(YL15)

YKL180W
RPL17A
Ribosomal protein L17A (L20A) (YL17)

YJL177W
RPL17B
Ribosomal protein L17B (L20B) (YL17)

YNL301C
RPL18B
Ribosomal protein L18B (rp28B)

YBL027W
RPL19B
Ribosomal protein L19B (YL14) (L23B)

(rp15L)

YGL135W
RPL1B
Ribosomal protein L1B

YMR242C
RPL20A
Ribosomal protein L20A (L18A)

YBR191W
RPL21A
Ribosomal protein L21A

YLR061W
RPL22A
Ribosomal protein L22A (L1c) (rp4) (YL31)

YGR148C
RPL24B
Ribosomal protein L24B (rp29) (YL21)

(L30B)

YLR344W
RPL26A
Ribosomal protein L26A (L33A) (YL33)

YFR031C-A
RPL2A
Ribosomal protein L2A (L5A) (rp8) (YL6)

YOR063W
RPL3
Ribosomal protein L3 (rp1) (YL1)

YGL030W
RPL30
Large ribosomal subunit protein L30 (L32)

(rp73) (YL38)

YDL075W
RPL31A
Ribosomal protein L31A (L34A) (YL28)

YBL092W
RPL32
Ribosomal protein L32

YPL143W
RPL33A
Ribosomal protein L33A (L37A) (YL37)

(rp47)

YOR234C
RPL33B
Ribosomal protein L33B (L37B) (rp47)

(YL37)

YDL191W
RPL35A
Ribosomal protein L35A

YMR194W
RPL36A
Ribosomal protein L36A (L39) (YL39)

YJR094W-A
RPL43B
Ribosomal protein L43B

YBR031W
RPL4A
Ribosomal protein L4A (L2A) (rp2) (YL2)

YDR012W
RPL4B
Ribosomal protein L4B (L2B) (rp2) (YL2)

YPL13LW
RPL5
Ribosomal protein L5 (L1a) (YL3)

YML073C
RPL6A
Ribosomal protein L6A (L17A) (rp18)

(YL16)

YLR448W
RPL6B
60S ribosomal subunit protein L6B (L17B)

(rp18) (YL16)

YPL198W
RPL7B
Ribosomal protein L7B (L6B) (rp11) (YL8)

YHL033C
RPL8A
Ribosomal protein L8A (rp6) (YL5) (L4A)

YLL045C
RPL8B
Ribosomal protein L8B (L4B) (rp6) (YL5)

YGL147C
RPL9A
Ribosomal protein L9A (L8A) (rp24) (YL11)

YHR027C
RPN1
Subunit of 26S Proteasome (PA700 subunit)

YHR200W
RPN10
homolog of the mammalian S5a protein,

component of 26S proteasome

YFR004W
RPN11
Similar to S. pombe PAD1 gene product

YFR052W
RPN12
cytoplasmic 32-34 kDa protein

YIL075C
RPN2
RPN2p is a component of the 26S

proteosome

YER021W
RPN3
component of the regulatory module of the

26S proteasome, homologous to human p58

subunit

YDL147W
RPN5
Subunit of the regulatory particle of the

proteasome

YDL097C
RPN6
Subunit of the regulatory particle of the

proteasome

YPR108W
RPN7
Subunit of the regulatory particle of the

proteasome

YOR261C
RPN8
Subunit of the regulatory particle of the

proteasome

YDR427W
RPN9
Subunit of the regulatory particle of the

proteasome

YLR340W
RPP0
60S ribosomal protein P0 (A0) (L10E)

YOL039W
RPP2A
60S acidic ribosomal protein P2A (L44)

(A2) (YP2alpha)

YDR382W
RPP2B
Ribosomal protein P2B (YP2beta) (L45)

YGR214W
RPS0A
Ribosomal protein S0A

YOR293W
RPS10A
Ribosomal protein S10A

YBR048W
RPS11B
Ribosomal protein S11B (S18B) (rp41B)

(YS12)

YOR369C
RPS12
40S ribosomal protein S12

YDR064W
RPS13
Ribosomal protein S13 (S27a) (YS15)

YCR031C
RPS14A
Ribosomal protein S14A (rp59A)

YOL040C
RPS15
40S ribosomal protein S15 (S21) (rp52)

(RIG protein)

YDL083C
RPS16B
Ribosomal protein S16B (rp61R)

YDR447C
RPS17B
Ribosomal protein S17B (rp51B)

YDR450W
RPS18A
Ribosomal protein S18A

YNL302C
RPS19B
Ribosomal protein S19B (rp55B) (S16aB)

(YS16B)

YLR441C
RPS1A
Ribosomal protein S1A (rp10A)

YML063W
RPS1B
Ribosomal protein S1B (rp10B)

YJL190C
RPS22A
Ribosomal protein S22A (S24A)

(rp50) (YS22)

YLR367W
RPS22B
Ribosomal protein S22B (S24B)

(rp50) (YS22)

YER074W
RPS24A
40S ribosomal protein S24A

YGR027C
RPS25A
Ribosomal protein S25A (S31A)

(rp45) (YS23)

YNL178W
RPS3
Ribosomal protein S3 (rp13) (YS3)

YLR287C-A
RPS30A
Ribosomal protein S30A

YHR203C
RPS4B
Ribosomal protein S4B (YS6) (rp5)

(S7B)

YJR123W
RPS5
Ribosomal protein S5 (S2) (rp14) (YS8)

YBR181C
RPS6B
40S ribosomal gene product S6B (S10B)

(rp9) (YS4)

YOR096W
RPS7A
Ribosomal protein S7A (rp30)

YNL096C
RPS7B
Ribosomal protein S7B (rp30)

YBL072C
RPS8A
Ribosomal protein S8A (S14A)

(rp19) (YS9)

YPL081W
RPS9A
Ribosomal protein S9A (S13) (rp21) (YS11)

YBR189W
RPS9B
Ribosomal protein S9B (S13) (rp21) (YS11)

YKL145W
RPT1
putative ATPase, 26S protease subunit

component

YDL007W
RPT2
(putative) 26S protease subunit

YOR117W
RPT5
26S protease regulatory subunit

YGL048C
RPT6
ATPase

YGL244W
RTF1
Nuclear protein

YLR180W
SAM1
S-adenosylmethionine synthetase

YDR502C
SAM2
S-adenosylmethionine synthetase

YAL005C
SSA1
Heat shock protein of HSP70 family,

cytoplasmic

YLL024C
SSA12
member of 70 kDa heat shock protein

family

YNL209W
SSB2
Heat shock protein of HSP70 family,

homolog of SSB1

YPL106C
SSE1
HSP70 family member, highly homologous

to Ssa1p and Sse2p

YLR150W
STM1
gene product has affinity for quadruplex

nucleic acids

YDR212W
TCP1
chaperonin subunit alpha

YGR192C
TDH3
Glyceraldehyde-3-phosphate

dehydrogenase 3

YBR118W
TEF2
translational elongation factor EF-1 alpha

YOL055C
THI20
THI for thiamine metabolism. Transcribed in

the presence of low level of thiamine

(10-8M) and turned off in the presence of

high level (10-6M) of thiamine. Under the

positive control of THI2 and THI3.

YDR050C
TPI1
triosephosphate isomerase

YJL130C
URA2
carbamoyl-phophate synthetase, aspartate

transcarbamylase, and glutamine

amidotransferase

YDL058W
USO1
Integrin analogue gene

YBL047C
YBL047C
USO1 homolog (S. cerevisiae), cytoskeletal-

related transport protein, Ca++ binding

YBL104C
YBL104C

YDR128W
YDR128W

YDR279W
YDR279W

YLR249W
YEF3
EF-3 (translational elongation factor 3)

YHL023C
YHL023C

YHR111W
YHR111W
moeB, thiF, UBA1

YMR247C
YMR247C

YOL138C
YOL138C

YPL110C
YPL110C

[0367]

8

TABLE 6

Excluded Ty protein gene identification numbers

7839187
7839173
6322010
7839201

6322347
7839155
7839171
6319369

7839188
7839156
7839205
6323688

7839162
6319468
7839159
6319467

7839207
7839160
7839195
6319485

7839180
7839194
6319486
6323597

6323689
6321110
7839164
7839199

6323695
6321547
6323601
7839185

6323694
6322486
6319324
2499832

2120056
141477
1323026
1323026

2499832
808856

[0368]

9

TABLE 7

Hypothetical proteins identified by HMS-PCI

ORF Name
Description
Gene

Q0032
questionable ORF

Q0092
questionable ORF

YAL008w
hypothetical protein
FUN14

YAL017w
similarity to ser/thr protein kinases
FUN31

YAL019w
similarity to helicases of the SNF2/RAD54
FUN30

family

YAL027w
hypothetical protein

YAL036c
strong similarity to GTP-binding proteins
FUN11

YAL049c
weak similarity to Legionella small basic

protein sbpA

YAL056w
similarity to hypothetical protein YOR371c
GPE2

YAR003w
similarity to human RB protein binding protein
FUN16

YAR014c
similarity to hypothetical protein S. pombe
BUD14

YAR044w
similarity to human oxysterol binding protein
OSH1

(OSBP)

YAR060c
identical to hypothetical protein YHR212c

YAR073w
strong similarity to IMP dehydrogenases
IMD1

YBL004w
weak similarity to Papaya ringspot virus

polyprotein

YBL029w
hypothetical protein

YBL032w
weak similarity to hnRNP complex protein

homolog YBR233w

YBL036c
strong similarity to C. elegans hypothetical

protein

YBL044w
hypothetical protein

YBL046w
weak similarity to hypothetical protein

YOR054c

YBL047c
similarity to mouse eps15R protein
EDE1

YBL049w
strong similarity to hypothetical protein—

human

YBL051c
similarity to S. pombe Z66S68_C protein

YBL055c
similarity to hypothetical S. pombe protein

YBL064c
strong similarity to thiol-specific antioxidant

enzyme

YBL095w
similarity to C. albicans hypothetical protein

YBL104c
weak similarity to S. pombe hypothetical

protein SPAC12G12.01c

YBL108w
strong similarity to subtelomeric encoded

proteins

YBR014c
similarity to glutaredoxin

YBR025c
strong similarity to Ylflp

YBR028c
similarity to ribosomal protein kinases

YBR030w
weak similarity to regulatory protein MSR1P

YBR046c
similarity to zeta-crystallin
ZTA1

YBR056w
similarity to glucan 1,3-beta-glucosidase

YBR063c
hypothetical protein

YBR066c
weak similarity to A. niger carbon catabolite
NRG2

repressor protein

YBR094w
weak similarity to pig tubulin-tyrosine ligase

YBR108w
weak similarity to R. norvegicus atrophin-1

related protein

YBR139w
strong similarity to carboxypeptidase

YBR150c
weak similarity to transcription factors
TBS1

YBR155w
weak similarity to stress-induced STI1P
CNS1

YBR158w
weak similarity to TRCDSEMBL:AF176518_1
CST13

F-box protein FBL2; human

YBR175w
similarity to S. pombe beta-tranaducin

YBR184w
hypothetical protein

YBR187w
similarity to mouse putative tranamembrane

protein FT27

YBR203w
hypothetical protein

YBR223c
hypothetical protein

YBR225w
hypothetical protein

YBR227c
similarity to E. coli ATP-binding protein clpX
MCX1

YBR228w
similarity to hypothetical Athaliana protein
SLX1

YBR239c
weak similarity to transcription factor PUT3P

YBR242w
strong similarity to hypothetical protein

YGL101w

YBR245c
strong similarity to D. melanogaster iswi
ISW1

protein

YBR246w
similarity to TREMBL:SPCC18_15

hypothetical protein, S. pombe

YBR259w
weak similarity to ‘BH1924’, sugar transport

system; Bacillus halodurans

YBR260c
similarity to C. elegans GTPase-activating
RGD1

protein

YBR264c
similarity to GTP-binding proteins
YPT10

YBR267w
similarity to hypothetical protein YLR3B7c

YBR269c
weak similarity to ‘cpa’, phospholipase C,

Clostridium perfringens

YBR270c
strong similarity to hypothetical protein

YJL058c

YBR280c
similarity to hypothetical protein S. pombe

YBR281c
similarity to hypothetical protein YFR044c

YCL010c
strong similarity to Saccharomyces pastorianus

hypothetical protein LgYCL010c

YCL039w
similarity to TUP1P general repressor of RNA

polymerase II transcription

YCL048w
strong similarity to sporulation-specific protein

SPS2P

YCL049c
similarity to unknown protein; S. pastorianus

YCL059c
strong similarity to fission yeast rev interacting
KRR1

protein mis3

YCL061c
similarity to URK1
MRC1

YCR001w
weak similarity to chloride channel proteins

YCR009c
similarity to human amphiphysin and RVS167P
RVS161

YCR030c
weak similarity to S. pombe hypothetical

protein SPBC4C3.06

YCR033w
similarity to nuclear receptor co-repressor N-Cor

YCR068w
similarity to starvation induced pSI-7 protein of
CVT17

C. fluvum

YCR076c
weak similarity to latent transforming growth

factor beta binding protein 3′ H. sapiens

YCR079w
weak similarity to A. thaliana protein

phosphatase 2C

YCR087w
questionable ORF

YCR099c
strong similarity to PEP1P, VTH1P and

VTH22p

YCR105w
strong similarity to alcohol dehydrogenases

YCR106w
similarity to transcription factor

YDL001w
similarity to hypothetical protein YFR048w,

YDR282c and S. pombe hypothetical protein

SPAC12G12.14

YDL019c
similarity to SWHIP
OSH2

YDL025c
similarity to probable protein kinase NPR1

YDL027c
weak similarity to hypothetical protein

Methanococcus jannaschii

YDL033c
similarity to H. influenzae hypothetical protein

H10174

YDL060w
similarity to C. elegans hypothetical protein
TSR1

YDL063c
weak similarity to human estrogen-responsive

finger protein

YDL074c
weak similarity to spindle pole body protein
BRE1

NUF1

YDL086w
similarity to hypothetical Synechocystis protein

YDL100c
similarity to E. coli arsenical pump-driving

ATPase

YDL113c
similarity to hypothetical protein YDR425w

YDL114w
weak similarity to Rhizobium nodulation

protein nodG

YDL117w
similarity to hypothetical S. pombe protein,
CYK3

protein possibly involved in cytokinesis

YDL119c
similarity to bovine Graves disease carrier

protein

YDL121c
hypothetical protein

YDL124w
similarity to aldose reductases

YDL129w
hypothetical protein

YDL156w
weak similarity to Pas7p

YDL172c
questionable ORF

YDL175c
weak similarity to cellular nucleic acid binding

proteins

YDL193w
similarity to N. crassa hypothetical 32 kDa

protein

YDL201w
strong similarity to probable methyltransferase

related protein Neurospora crassa

YDL204w
similarity to hypothetical protein YDR233c

YDL206w
weak similarity to transporter proteins

YDL213c
weak similarity to potato small nuclear
FYV14

ribonucleoprotein U2B and human splicing

factor homolog

YDL214c
strong similarity to putative protein kinase
PRR2

NPR1

YDL224c
strong similarity to WHI3 protein
WHI4

YDL239c
hypothetical protein
ADY3

YDL244w
strong similarity to THI5P, YJRI56c,
THI13

YNL332w and A. parasiticus, S. pombe

NMT1 protein

YDL248w
strong similarity to subtelomeric encoded
COS7

proteins

YDR018c
strong similarity to hypothetical protein

YBR042c

YDR032c
strong similarity to S. pombe obr1 brefeldin
PST2

A resistance protein

YDR036c
similarity to enoyl CoA hydratase

YDR049w
similarity to C. elegans K06H7.3 protein

YDR055w
strong similarity to SPS2 protein
PST1

YDR063w
weak similarity to glia maturation factor beta

YDR071c
similarity to G. aries arylalkylamine

N-acetyltransferase

YDR091c
strong similarity to human RNase L inhibitor
RLI1

and M. jannaschii ABC transporter protein

YDR093w
similarity to P. falciparum ATPase 2

YDR101c
weak similarity to proliferation-associated

protein

YDR102c
hypothetical protein

YDR106w
similarity to Actin proteins
ARP10

YDR116c
similarity to bacterial ribosomal Li proteins

YDR119w
similarity to B. subtilis tetracyclin resistance

YDR124w
hypothetical protein

YDR125w
weak similarity to SEC27P, YMR131c and

human retinoblastoma-binding protein

YDR131c
similarity to hypothetical protein YJL149w

YDR141c
strong similarity to Emericella nidulans
DOP1

developmental regulatory gene, dopey (dopA)

YDR152w
weak similarity to C. elegans hypothetical

protein CET26E3

YDR161w
weak similarity to S. pombe protein of
TCI1

unknown functionSPBC16D10.01c

YDR163w
weak similarity to S. pombe hypothetical

protein

YDR165w
weak similarity to hypothetical C. elegans

protein

YDR186c
hypothetical protein

YDR196c
similarity to C. elegans hypothetical protein

T05G5.5

YDR198c
similarity to hypothetical protein S. pombe

YDR200c
similarity to hypothetical protein YLR238w

similarity to A. eutrophus cation efflux system

membrane protein czcD, rat zinc transport

YDR205w
protein ZnT
MSC2

YDR214w
similarity to hypothetical protein YNL2S1w

YDR219c
hypothetical protein

YDR229w
similarity to hypothetical protein N. crassa

YDR233c
similarity to hypothetical protein YDL204w

YDR239c
hypothetical protein

YDR247w
strong similarity to SKS1P

YDR255c
weak similarity to hypothetical S. pombe

hypothetical protein SPBC29A3

YDR266c
similarity to hypothetical C. elegans protein

YDR267c
weak similarity to human TAFII100 and other

WD-40 repeat containing proteins

YDR274c
hypothetical protein

YDR275w
weak similarity to YOR042w

YDR279w
hypothetical protein

YDR282c
similarity to hypothetical protein YDL001w, YFR048w

and S. pombe hypothetical protein

SPAC12G12.14

YDR287w
similarity to inositolmonophosphatases

YDR295c
weak similarity to USO1P, YPR179c and fruit
PLO2

fly tropomyosin

YDR303c
similarity to transcriptional regulator proteins
RSC3

YDR306c
weak similarity to S. pombe hypothetical

protein SPAC6F6

YDR316w
similarity to hypothetical ubiquitin system

protein S. pombe

YDR324c
weak similarity to beta transducin from S. pombe

and other WD-40 repeat containing proteins

YDR326c
strong similarity to YHR080c, similarity to

YFL042c and YLR072w

YDR332w
similarity to E. coli hypothetical protein

and weak similarity to RNA helicase

MSS116/YDR194c

YDR339c
weak similarity to hypothetical protein

YOR004w

YDR344c
hypothetical protein

YDR359c
weak similarity to human trichohyalin
VID21

YDR361c
similarity to hypothetical protein S. pombe
BCP1

YDR365c
weak similarity to Streptococcus M protein

YDR368w
strong similarity to members of the aldo/keto

reductase family YPR1

YDR372c
similarity to hypothetical S. pombe protein

YDR3S0w
similarity to PDC6P, THI3P and to pyruvate
ARO10

decarboxylases

YDR393w
weak similarity to rabbit trichohyalin
SHE9

YDR395w
similarity to human KIAA0007 gene

YDR412w
questionable ORF

YDR449c
similarity to hypothetical protein S. pombe

YDR452w
similarity to human sphingomyelin
PHM5

phosphodiesterase (PIR:S06957)

YDR453c
strong similarity to thiol-specific antioxidant

proteins

YDR459c
weak similarity to YNL326c

YDR466w
similarity to ser/thr protein kinase

YDR452c
hypothetical protein

YDR496c
similarity to hypothetical human and

C. elegans
proteins

YDR506c
similarity to FET3, YFL041w and

E. floriforme
diphenol oxidase

YDR516c
strong similarity to glucokinase

YDR527w
weak similarity to Plasmodium yoelii

rhoptry protein

YEL015w
weak similarity to SPA2P

YEL018w
weak similarity to RAD50P

YEL023c
similarity to hypothetical protein PA2063—

Pseudomonas aeruginosa

YEL025c
hypothetical protein
SRI1

YEL038w
similarity to K. oxytoca enolase-
UTR4

phosphatase E-1

YEL064c
similarity to YBL089w

YEL070w
strong similanty to E. coli D-mannonate

oxidoreductase

YEL077c
strong similarity to subtelomeric encoded

proteins

YER002w
weak similarity to chicken microfibril-

associated protein

YER006w
similarity to P. polycephalum myosin-related

protein mlpA

YER010c
similarity to L. pneumophila dlpA protein

YER019w
weak similarity to human and mouse neutral
ISC1

sphingomyelinase

YER030w
similarity to mouse nucleolin

YER036c
strong similarity to members of the ABC
KRE30

transporter family

YER041w
weak similarity to DNA repair protein RAD2P
YEN1

and Dsh1p

YER049w
strong similarity to hypothetical S. pombe

protein YER049W

YER066c-a
hypothetical protein

YER066w
strong similarity to cell division control

protein CDC4P

YER067w
strong similarity to hypothetical protein

YIL057c

YER077c
hypothetical protein

YER078c
similarity to E. coli X-Pro aminopeptidase II

YER080w
hypothetical protein

YER082c
similarity to M. sexta steroid regulated MNG10
KRE31

protein

YER083c
hypothetical protein

YER084w
questionable ORF

YER087w
similarity to E. coli prolyl-tRNA synthetase

YER093c
weak similarity to S. epidermidis PepB protein

YER124c
weak similarity to Dictyostelium WD40 repeat

protein 2

YER126c
weak similarity to E. coli colicin N
KRE32

YER130c
similarity to MSN2P and weak similarity to

MSN4P

YER140w
similarity to PIR:T39406 hypothetical protein

S. pombe

YER158c
weak similarity to AFR1P

YER166w
similarity to ATPase P. falciparum ATPase 2

YER182w
similarity to hypothetical protein

SPAC3A12.08—S. pombe

YER184c
similarity to multidrug resistance proteins

PDR3P and PDR1P

YER185w
strong similarity to Rtm1p

YFL006w
similarity to hypothetical protein

TRCDSEMBL:AB024034_15 A. thaliana

YFL007w
weak similarity to Mms19p
BLM3

YFL013c
weak similarity to Dictyostelium protein kinase
IES1

YFL024c
weak similarity to YMR164c and GAL11P
EPL1

YFL027c
weak similarity to P. falciparum Pfmdr2

protein

YFL030w
similarity to several transaminases

YFL034w
similarity to hypothetical S. pombe protein

and to C. elegans F35D11 protein

YFL042c
similarity to hypothetical protein YLR072w

YFL054c
similarity to channel proteins

YFR001w
weak similarity to rabbit triadin SPP41P
L0C1

YFR003c
strong similarity to hypothetical protein

SPAC6B12.13—S. pombe

YFR008w
weak similarity to human centromere protein E

YFR016c
similarity to mammalian neurofilament proteins

and to Dictyostelium protein kinase

YFR017c
hypothetical protein

YFR021w
similarity to hypothetical protein YPL100w
NMR1

YFR024c-a
similarity to Acanthamoeba myosin heavy

chain IC and weak similarity to other myosin

class I heavy c

YFR039c
similarity to hypothetical protein YGL228w

YFR044c
similarity to hypothetical protein YBR281c

YGL004c
weak similarity to TUP1P

YGL020c
weak similarity to

TRCDSEMBL:SPBC543_10 putative

coiled-coil protein S. pombe

YGL037c
similarity to PIR:B70386 pyrazinamidase/
PNC1

nicotinamidase—Aquifex aeolicus

YGL057c
hypothetical protein

YGL059w
similarity to rat branched-chain alpha-ketoacid

dehydrogenase kinase

YGL060w
strong similarity to hypothetical protein

YBR216c

YGL068w
strong similarity to Cricetus mitochrondial

ribosomal L12 protein

YGL081w
hypothetical protein

YGL083w
weak similarity to bovine rhodopsin kinase and
SCY1

to YGR052w

YGL096w
similarity to copper homeostasis protein
TOS8

CUP9P

YGL099w
similarity to putative human GTP-binding
KRE35

protein MMR1

YGL101w
strong similarity to hypothetical protein

YBR242w

YGL104c
similarity to glucose transport proteins

YGL110c
similarity to hypothetical protein

SPCC1906.02c S. pombe

YGL111w
weak similarity to hypothetical protein

S. pombe

YGL113w
weak similarity to YOR165w
SLD3

YGL117w
hypothetical protein

YGL121c
hypothetical protein

YGL129c
similarity to S. pombe pl hypothetical protein
RSM23

SPBC29A3.15C—putative mitochondrial

function

YGL131c
weak similarity to S. pombe hypothetical

protein C3H1.12C

YGL140c
weak similarity to Lactobacillus putative histidine

protein kinase SppK

YGL146c
hypothetical protein

YGL150c
similarity to SNF2P and human SNF2alpha
INO80

YGL174w
weak similarity to C. elegans hypothetical
BUD13

protein R08D7.1

YGL179c
strong similarity to PAK1P, ELM1P and
TOS3

KIN82P

YGL184c
strong similarity to Emericella nidulans and
STR3

similarity to other cystathionine beta-lyase and

CYS3P

YGL220w
weak similarity to V. alginolyticus bolA protein

YGL222c
weak similarity to EDC2
EDC1

YGL227w
weak similarity to human RANBPM
VID30

NP_005484.1

YGL228w
similarity to hypothetical protein YFR039c
SHE10

YGL245w
strong similanty to glutamine—tRNA ligase

YGL246c
weak similarity to C. elegans dom-3 protein
RAI1

YGR002c
similarity to hypothetical S. pombe protein

YGR004w
strong similarity to hypothetical protein

YLR324w

YGR016w
weak similarity to M. jannaschii hypothetical

protein MJ1317

YGR017w
weak similarity to

TRCDSEMBL:AC006418_11 A. thaliana

YGR021w
similarity to M. leprae yfcA protein

YGR033c
weak similarity to

TRCDSEMBLNEW:AP002861_10

Oryza sativa

YGR042w
weak similarity to TRCDSEMBL:CH20111_1

Troponin-I; Clupea harengus

YGR043c
strong similarity to transaldolase

YGR052w
similarity to ser/thr protein kinases

YGR054w
similarity to C. elegans E04D5.1 protein

YGR066c
similarity to hypothetical protein YBR105c

YGR067c
weak similarity to transcription factors

YGR073e
questionable ORF

YGR077c
similarity to Hansenula polymorpha PER1
PEX8

protein and weak similanty to Pichia pastoris

PER3 protein

YGR086c
strong similarity to hypothetical protein

YPL004c

YGR090w
similarity to PIR:T40678 hypothetical protein

SPBC776.08c S. pombe

YGR103w
similarity to zebrafish essential for embryonic

development gene pescadillo

YGR110w
weak similarity to YLR099c and YDR125c

YGR111w
weak similarity to mosquito carboxylesterase

YGR128c
hypothetical protein

YGR130c
weak similarity to myosin heavy chain proteins

YGR134w
hypothetical protein
CAF130

YGR136w
weak similarity to chicken growth factor

receptor-binding protein GRB2 homolog

YGR145w
similarity to C. elegans hypothetical

protein

YGR150c
similarity to PIR:T39838 hypothetical protein

SPBC19G7.07c S. pombe

YGR154c
strong similarity to hypothetical proteins

YKR076w and YMR251w

YGR161c
hypothetical protein

YGR165w
similarity to PIR:T39444 hypothetical protein

SPBC14C8.16c S. pombe

YGR169c
similarity to RIB2P

YGR173w
strong similarity to human GTP-binding protein

YGR187c
weak similarity to human HMG1P and HMG2P
HGH1

YGR196c
weak similarity to Tetrahymena acidic
FYV8

repetitive protein ARP1

YGR198w
weak similarity to PIR:T38996 hypothetical

protein SPAC637.04 S. pombe

YGR200c
weak similarity to rape guanine nucleotide
ELP2

regulatory protein

YGR205w
similarity to S. pombe hypothetical protein

D89234

YGR210c
similarity to M. jannaschii GTP-binding protein

and to M. caprtcolum hypothetical protein

SGC3

YGR223c
weak similarity to hypothetical protein

YFR021w

YGR235c
hypothetical protein

YGR243w
strong similarity to hypothetical protein

YHR162w

YGR250c
weak similarity to human cleavage stimulation

factor 64K chain

YGR262c
weak similarity to protein kinases and M.
BUD32

jannaschii
O-sialoglycoprotein endopeptidase

homolog

YGR263c
weak similarity to E. coli lipase like enzyme

YGR266w
hypothetical protein

YGR271w
strong similarity to S. pombe RNA helicase
SLH1

YGR278w
similarity to C. elegans LET-858

YGR279c
similarity to glucanase
SCW4

YGR280c
weak similarity to CBF5P

YGR296w
strong similarity to YPL283c; YNL339c and
YRF1-3

other Y encoded proteins

YHL010c
similarity to C. elegans hypothetical protein,

homolog to human breast cancer-associated

protein BRAP

YHL013c
similarity to C. elegans hypothetical protein

F21D5.2

YHL014c
similarity to E. coli GTP-binding protein
YLF2

YHL017w
strong similarity to PTM1P

YHL023c
weak similarity to TRCDSEMBL:SPBC543_4

hypothetical protein S. pombe

YHL026c
similarity to PIR:T41446 conserved

hypothetical protein SPCC594.02c S. pombe

YHL035c
similarity to multidrug resistance proteins

YHL039w
weak similarity to YPL208w

YHR001w
similarity to KES1P
OSH7

YHR002w
similarity to bovine mitochondrial carrier

protein/Grave's disease carrier protein

YHR009c
similarity to S. pombe hypothetical protein

YHR011w
strong similarity to seryl-tRNA synthetases
DIA4

YHR016c
strong similarity to hypothetical protein
YSC84

YFR024c-a

YHR020w
strong similarity to human glutamyl-prolyl-

tRNA synthetase and fruit fly multifunctional

aminoacyl-t

YHR022c
weak similarity to ras-related protein

YHR033w
strong similarity to glutamate 5-kinase

YHR035w
weak similarity to human SEC23 protein

YHR040w
weak similarity to HIT1P

YHR045w
hypothetical protein

YHR046c
similarity to inositolmonophosphatases

YHR052w
weak similarity to P. yoelii rhoptry protein

YHR056c
strong similarity to YHR054c
RSC30

YHR059w
weak similarity to Ustilago hordei B east
FYV4

mating protein 2

YHR063c
weak similarity to translational activator CBS2
PAN5

YHR070w
strong similarity to N. crassa met-10+ protein
TRM5

YHR073w
similarity to OSH1P, YDL019c and mammalian
OSH3

oxysterol-binding protei

YHR074w
weak similarity to B. subtilis spore outgrowth
QNS1

factor B

YHR076w
weak similarity to C. elegans hypothetical

protein CEW09D10

YHR080c
similarity to hypothetical protein YDR326c,

YFL042c and YLR072w

YHR087w
weak similarity to PIR:T50363 hypothetical

protein SPBC21C3.19 S. pombe

YHR088w
similarity to hypothetical protein YNL07Sw
RPF1

YHR098c
similarity to human hypothetical protein
SFB3

YHR100c
strong similarity to PIR:T48794 hypothetical

protein Neurospora crassa

YHR105w
weak similarity to MVP1P

YHR111w
similarity to molybdopterin biosynthesis

proteins

YHR112c
similarity to cystathionine gamma-synthases

YHR113w
similarity to vacuolar aminopeptidase Ape1p

YHR114w
similarity to S. pombe hypothetical protein
BZZ1

and human protein-tyrosine kinase fer

YHR115c
strong similarity to hypothetical protein

YNL116w

YHR122w
similarity to hypothetical C. elegans protein

F45G2.a

YHR149c
similarity to hypothetical protein YGR221c

YHR169w
strong similarityto DRS1P and other probable
DBP8

ATP-dependent RNA helicases

YHR177w
weak similarity to S. pombe PAC2 protein

YHR182w
weak similarity to PIR:S58162 probable Rho

GTPase protein S. pombe

YHR186c
similarity to C. elegans hypothetical protein

C10C5.6

YHR188c
similarity to hypothetical C. elegans proteins
GPI16

F17c11.7

YHR196w
weak similarity to YDR398w

YHR197w
weak similarity to PIR:T22172 hypothetical

protein F44E5.2 C. elegans

YHR199c
strong similarity to hypothetical protein

YHR198c

YHR209w
similarity to hypothetical protein YER175c

YHR214w-a
strong similarity to hypothetical protein

YAR068w

YIL005w
similarity to protein disulfide isomerases

YIL007c
similarity to C. elegans hypothetical protein

YIL017c
similarity to S. poinbe SPAC26H5.04 protein
VID28

of unknown function

YIL028w
hypothetical protein

YIL037c
weak similarity to C. elegans F26G1.6 protease
PRM2

YIL055c
hypothetical protein

YIL077c
hypothetical protein

YIL079c
strong similarity to hypothetical protein

YDL175c

YIL091c
weak similarity to SPT5P

YIL093c
weak similarity to S. pombe hypothetical
RSM25

protein SPBC16A3

YIL097w
weak similanty to erythroblast macrophage
FYV10

protein EMP Mus musculus

YIL104c
similarity to hypothetical S. pombe protein

YIL105c
weak similarity to probable transcription factor

ASK10P

YIL108w
similarity to hypothetical S. pombe protein

YIL112w
similarity to ankyrin and coiled-coil proteins

YIL113w
strong similarity to dual-specificity phosphatase

MSG5P

YIL117c
similarity to hypothetical protein YNL058c
PRM5

YIL120w
similarity to antibiotic resistance proteins
QDR1

YIL137c
similarity to M. musculus aminopeptidase

YIL164c
strong similarity to nitrilases, putative
NIT1

pseudogene

YIR00Lc
similarity to D. melanogaster RNA binding
SGN1

protein

YIR003w
weak similarity to mammalian neurofilament

triplet H proteins

YIR005w
similarity to RNA-binding proteins
IST3

YIR007w
hypothetical protein

YIR035c
similarity to human corticosteroid

11-beta-dehydrogenase

YJL019w
weak similarity to hypothetical protein

C. elegans

YJL020c
similarity to S. pombe hypothetical protein
BBC1

SPAC23A1.16

YJL038c
strong similarity to hypothetical protein

YJL037w

YJL045w
strong similarity to succinate dehydrogenase

flavoprotein

YJL047c
weak similarity to CDC53P
RTT101

YJL051w
hypothetical protein

YJL066c
hypothetical protein
MPMt

YJL068c
strong similarity to human esterase D

YJL069c
similarity to C. elegans hypothetical protein

YJL070c
similarity to AMP deaminases

YJL073w
similarity to heat shock proteins
JEM1

YJL082w
strong similarity to hypothetical protein
IML2

YKR018c

YJL084c
similarity to hypothetical protein YKR021w

YJL105w
similarity to hypothetical protein YKR029c

YJL107c
similarity to hypothetical S. pombe protein

YJL109c
weak similarity to ATPase DRS2P

YJL122w
weak similarity to dog-fish transition protein 52

YJL132w
weak similarity to human phospholipase D

YJL149w
similarity to hypothetical protein YDR131c

YJL181w
similarity to hypothetical protein YJR030c

YJL204c
weak similarity to TOR2P

YJL207c
weak similarity to rat omega-conotoxin-

sensitive calcium channel alpha-1 subunit rbB-I

YJL211c
questionable ORF

YJR011c
hypothetical protein

YJR024c
weak similarity to C. elegans Z49131_E

ZC373.5 protein

YJR041c
weak similarity to hypothetical protein

SPAC2G11.02 S. pombe

YJR054w
similarity to hypothetical protein YML047c

YJR061w
similarity to MNN4P

YJR070c
similarity to C. elegans hypothetical protein

C14A4.1

YJR072c
strong similarity to C. elegans hypothetical

protein and similarity to YLR243w

YJR078w
similarity to mammalian indoleamine

2,3-dioxygenase

YJR080c
hypothetical protein

YJR087w
questionable ORF

YJR100c
weak similarity to BUD3P

YJR101w
weak similarity to superoxide dismutases
RSM26

YJR105w
strong similarity to human adenosine kinase
ADO1

YJR110w
similarity to human myotubularmn

YJR119c
similarity to human retinoblastoma binding

protein 2

YJR126c
similarity to human prostate-specific membrane

antigen and transferrin receptor protein

YJR129c
weak similarity to hypothetical protein

YNL024c

YJR134c
similarity to paramyosin, myosin
SGM1

YJR138w
similarity to C. elegans hypothetical protein
IML1

T0BA11.1

YJR141w
weak similarity to hypothetical protein

SPBC1734.10c S. pombe

YJR149w
similarity to 2-nitropropane dioxygenase

YJR151c
similarity to mucus proteins, YKL224c, Sta1p
DAN4

YKL010c
similarity to rat ubiquitin ligase Nedd4
UFD4

YKL014c
similarity to hypothetical protein

SPCC14G10.02 S. pombe

YKL018w
similarity to C. elegans hypothetical protein

YKL034w
weak similarity to YOL013c

YKL036c
questionable ORF

YKL047w
hypothetical protein

YKL054c
similarity to glutenin, high molecular weight
VID31

chain proteins and SNF5P

YKL056c
strong similarity to human 1gB-dependent

histamine-releasing factor

YKL075c
hypothetical protein

YKL082c
weak similarity to C. elegans hypothetical

protein

YKL088w
similarity to C. tropicalis hal3 protein, to

C-term of SIS2P and to hypothetical protein

YOR054c

YKL095w
similarity to C. elegans hypothetical proteins
YJU2

YKL099c
similarity to C. elegans hypothetical proteins

C18G6.06 and C16C10.2

YKL105c
similarity to YMR086w

YKL116c
similarity to rat SNF1, C. elegans unc-51,
PRR1

DUN1P and other protein seine kinases

YKL120w
similarity to mitochondrial uncoupling proteins
OAC1

(MCF)

YKL121w
strong similarity to YMR102c

YKL133c
similarity to hypothetical protein YMR115w

YKL155c
similarity to S. pombe SPAC1420.04c putative
RSM22

cytochrome c oxidase assembly protein

YKL161c
strong similarity to ser/thr-specific protein
(MLP1)

kinase SLT2P

YKL179c
similarity to NUF1P

YKL189w
similarity to mouse hypothetical calcium-
HYM1

binding protein and D. melanogaster

Mo25 gene

YKL195w
similarity to rabbit histidine-rich calcium-

binding protein

YKL206c
hypothetical protein

YKL214c
weak similarity to mouse transcriptional

coactivator ALY

YKL215c
similarity to P. aeruginosa hyuA and hyuB

YKL218c
strong similarity to E. coli and H. influenzae
SRY1

threonine dehydratases

YKL222c
weak similarity to transcription factors,

similarity to finger proteins YOR162c,

YOR172w and YLR266c

YKR005c
hypothetical protein

YKR007w
weak similarity to Streptococcus protein M5

precursor

YKR017c
similarity to human hypothetical KIAA0161

protein

YKR018c
strong similarity to hypothetical protein

YJL082w

YKR020w
hypothetical protein

YKR029c
similarity to YJL105w and Lentinula MFBA

protein

YKR038c
similarity to QR17P

YKR046c
hypothetical protein

YKR051w
similarity to C. elegans hypothetical protein

YKR060w
similarity to hypothetical protein S. pombe

YKR064w
weak similarity to transcription factors

YKR065c
similarity to hypothetical protein S. pombe

YKR067w
strong similarity to SCT1P

YKR079c
similarity to S. pombe hypothetical protein

SPAC1D4.10

YKR081c
strong similarity to hypothetical protein

S. pombe

YKR090w
similarity to chicken Lim protein kinase and

Islet proteins

YKR096w
similarity to mitochondrial aldehyde

dehydrogenase Ald1p

YLL013c
similarity to Drosophila pumilio protein

YLL015w
similarity to YCF1P, YOR1P, rst organic anion
BPT1

transporter

YLL029w
similarity to M. jannaschii X-Pro dipeptidase

and S. pombe hypothetical protein

YLL034c
similarity to mammalian valosin

YLL038c
weak similarity to YJR125c and YDL161w
ENT4

YLL054c
similarity to transcription factor PIP2P

YLL063e
strong similarity to Gibberella zeae
AYT1

trichothecene 3-O-acetyltransferase

YLR002c
similarity to hypothetical C. elegans protein

YLR009w
similarity to ribosomal protein L24.e.B

YLR015w
weak similarity to S. pombe hypothetical
BRE2

protein SPBC13G1

YLR016c
weak similarity to

TRCDSEMBLNEW:AK022615_1 unnamed

ORF; Homo sapiens

YLR024c
similarity to ubiquitin—protein ligase UBR1P
UBR2

YLR035c
similarity to human mutL protein homolog,
MLH2

mouse PMS2, MLH1P and PMS1P

YLR062e
questionable ORF
BUD28

YLR063w
hypothetical protein

YLR070c
strong similarity to sugar dehydrogenases

YLR074c
weak similarity to human zinc finger protein
BUD20

YLR080w
strong similarity to EMP47P

YLR087c
weak similarity to hypothetical protein
CSF1

D. melanogaster

YLR097c
weak similarity to H. sapiens F-box protein

YLR106c
similarity to Kaposi's sarcoma-associated

herpes-like virus ORF73 homolog gene

YLR117c
strong similarity to Drosophila putative cell
CLF1

cycle control protein cm

YLR122c
hypothetical protein

YLR152c
similarity to YOR3165w and YNL095c

YLR154c
hypothetical protein

YLR177w
similarity to suppressor protein Psp5p

YLR179c
similarity to TFS1P

YLR183c
similarity to YDR501w
TOS4

YLR186w
strong similarity to S. pombe hypothetical
EMG1

protein C18C36.07C

YLR187w
similarity to hypothetical protein YNL278w

YLR193c
similarity to G. gallus pxt9 and MSF1P

YLR196w
similarity to human IEF SSP 9502 protein
PWP1

YLR199c
hypothetical protein

YLR205c
hypothetical protein

YLR211e
hypothetical protein

YLR215c
strong similarity to rat cell cycle progression
CDC123

related D123 protein

YLR219w
hypothetical protein
MSC3

YLR222c
similarity to DIP2P
CST29

YLR231c
strong similarity to rat kynureninase

YLR238w
similarity to YDR200c

YLR241w
similarity to hypothetical S. pombe

protein SPAC2G11.09

YLR243w
strong similarity to YOR262w

YLR247c
similarity to S. pombe rad8 protein and

RDH54P

YLR266c
weak similarity to transcription factors

YLR267w
hypothetical protein
BOP2

YLR270w
strong similarity to YOR173w

YLR271w
weak similarity to hypothetical protein

T04H1.5 C. elegans

YLR276c
similarity to YDL031w, MAK5P and RNA
DBP9

helicases

YLR282c
questionable ORF

YLR287c
weak similarity to S. pombe hypothetical

protein SPAC22E12

YLR289w
strong similarity to E. coli elongation
GUF1

factor-type GTP-hinding protein lepa

YLR320w
hypothetical protein

YLR323c
weak similarity to N. crassa uvs2 protein

YLR324w
strong similarity to YGR004w

YLR326w
hypothetical protein

YLR328w
strong similarity to YGR010w

YLR331c
questionable ORF

YLR349w
questionable ORF

YLR352w
hypothetical protein

YLR368w
weak similarity to Mus musculus F-box

protein FBA

YLR373c
similarity to hypothetical protein Ygr071cp
VID22

YLR381w
hypothetical protein

YLR386w
similarity to hypothetical S. pombe protein

YLR392c
hypothetical protein

YLR397c
strong similarity to CDC48
AFG2

YLR400w
hypothetical protein

YLR401c
similarity to A. brasilense nifR3 protein

YLR405w
similarity to A. brasilense nifR3 protein

YLR409c
strong similarity to S. pombe beta-transducin

YLR410w
strong similarity to S. pombe protein ASP1P
VIP1

YLR413w
strong similarity to YKL187c

YLR415c
questionable ORF

YLR419w
similarity to helicases

YLR421c
weak similarity to human 42K membrane
RPN13

glycoprotein

YLR422w
similarity to human DOCK180 protein

YLR424w
weak similarity to STU1P

YLR425w
similarity to GDP-GTP exchange factors
TUS1

YLR426w
weak similarity to 3-oxoacyl-[acyl-carrier-

protein] reductase from E. coli

YLR427w
weak similarity to human transcription

regulator Staf-5

YLR432w
strong similarity to IMP dehydrogenases, Pur5p
IMD3

and YML056c

YLR454w
similarity to YPR117w

YLR460c
similarity to C. carbonum toxD protein

YML002w
hypothetical protein

YML005w
similarity to hypothetical S. pombe protein

YML006c
hypothetical protein
GIS4

YML020w
hypothetical protein

YML023c
weak similarity to NMD2P

YML029w
hypothetical protein

YML034w
similarity to YDR458c
SRC1

YML036w
weak similarity to C. elegans hypothetical

protein CELW03F8

YML056c
strong similarity to IMP dehydrogenases
IMD4

YML059c
similarity to C. elegans ZK370.4 protein

YML068w
similarity to C. elegans hypothetical protein

YML072c
similarity to YOR3141c and YNL087w

YML076c
weak similarity to transcription factor

YML081w
strong similarity to ZMS1 protein

YML093w
similarity to P. falciparum liver stage

antigen LSA-1

YML111w
strong similarity to ubiquitination protein
BUL2

BUL1P

YML117w
similarity to YPL184c

YML128c
weak similarity to S. pombe SPBC365.12c
MSC1

protein of unknown function

YMR015w
similarity to tetratricopeptide-repeat protein

PAS10

YMR019w
weak similarity to YIL130w, PUT3P and
STB4

other transcription factors

YMR029c
weak similarity to human nuclear autoantigen

YMR030w
hypothetical protein

YMR049c
weak similarity to A. thaliana PRL1 protein
ERB1

YMR066w
hypothetical protein
SOV1

YMR068w
weak similarity to mouse transcription factor

NF-kappaB

YMR074c
strong similarity to hypothetical S. pombe

protein

YMR086w
similarity to YKLL05c

YMR093w
weak similarity to PWP2P

YMR099c
similarity to P. ciliare possible

apospory-associated protein

YMR102c
strong similarity to YKL121w

YMR135c
weak similarity to conserved hypothetical

protein S. pombe

YMR144w
weak similarity to MLP1P

YMR155w
weak similarity to E. coli hypothetical

protein f402

YMR172w
similarity to MSN1 protein
HOT1

YMR196w
strong similarity to hypothetical protein

Neurospora crassa

YMR206w
weak similarity to hypothetical protein

YNR014w

YMR207c
strong similarity to acetyl-CoA carboxylase
HFA1

YMR209c
similarity to conserved hypothetical protein

S. pombe

YMR223w
similarity to mouse deubiquitinating enzyme
UBP8

and UBP13P, UBP9, DOA4P

YMR226c
similarity to ketoreductases

YMR233w
strong similarity to YOR295w

YMR247c
similarity to hypothetical protein S. pombe

YMR250w
similarity to glutamate decarboxylases
GAD1

YMR251w
strong similarity to YKR076w and YGR154c

YMR259c
similarity to hypothetical protein S. pombe

YMR265c
weak similarity to hypothetical protein

S. pombe

YMR266w
similarity to A. thaliana hyp1 protein
RSN1

YMR278w
similarity to phosphomannomutases

YMR285c
similarity to CCR4P
NGL2

YMR289w
weak similarity to para-aminobenzoate synthase

component I (EC 4.1.3.-) Campylobacter jejuni

YMR291w
similarity to ser/thr protein kinase

YMR304w
similarity to human ubiquitin-specific protease
UBP15

YMR306w
similarity to 1,3-beta-glucan synthases
FKS3

YMR315w
similarity to hypothetical S. pombe protein

YMR316w
similarity to YOR385w and YNL165w
DIA1

YMR318c
strong similarity to alcohol-dehydrogenase

YMR323w
strong similarity to phosphopyruvate hydratases

YNL102c
strong similarity to mammalian ribosomal L7
RLP7

proteins

YNL004w
strong similarity to GBP2P
HRB1

YNL008c
similarity to YMR119w

YNL023e
similarity to D. melanogaster shuttle craft
FAP1

protein

YNL032w
similarity to YNL099c, YNL056w and
SIW14

YDR067c

YNL035c
similarity to hypothetical protein S. pombe

YNL040w
weak similarity to M. genitalium alanine—

tRNA ligase

YNL045w
strong similarity to human leukotriene-A4

hydrolase

YNL047c
similarity to probable transcription factor

ASK10P and hypothetical protein YPR115w, and

strong simi

YNL051w
weak similarity to hypothetical protein

Drosophila melanogaster

YNL056w
similarity to YNL032w and YNL099c

YNL063w
weak similarity to Mycoplasma

protoporphyrinogen
oxidase

YNL078w
hypothetical protein

YNL083w
weak similarity to rabbit peroxisomal

Ca-dependent solute carrier

YNL091w
similarity to chicken h-caldesmon, USO1P

and YKL201c

YNL094w
similarity to S. pombe hypothetical protein

YNL096c
strong similarity to ribosomal protein S7
RPS7B

YNL099c
similarity to YNL032w, YNL056w and

YDR067c

YNL107w
similarity to human AF-9 protein
YAF9

YNL108c
strong similarity to YOR110w

YNL109w
weak similarity to cytochrome-c oxidase

YNL110c
weak similarity to fruit fly RNA-binding

protein

YNL116w
weak similarity to RING zinc finger protein

from Gallus gallus

YNL123w
weak similarity to C. jejuni acme protease

YNL124w
similarity to hypothetical S. pombe protein

YNL127w
similarity to C. elegans hypothetical protein

YNL128w
weak similarity to tensin and to the mammalian
TEP1

tumor suppressor gene product

MMAC1/PTEN/TEP1

YNL132w
similarity to A. ambisexualis anthendiol
KRE33

steroid receptor

YNL134c
similarity to C. carbonum toxD gene

YNL144c
similarity to YHR131c

YNL157w
weak similarity to S. pombe hypothetical

protein SPAC10F6

YNL161w
strong similarity to U. maydis Ukc1p protein
CBK1

kinase

YNL166c
similarity to S. pombe SPBC1711.05
BNI5

serine-rich repeat protein of unknown function

YNL175c
similarity to S. pombe Rnp24p, NSR1P and
NOP13

human splicing factor

YNL180c
similarity to S. pombe CDC42P and other
RHO5

GTP-hinding proteins

YNL181w
similarity to hypothetical S. pombe protein

YNL182c
weak similarity to S. pombe hypothetical

protein

YNL201c
weak similarity to pleiotropic drug resistance

control protein PDR6

YNL207w
similarity to M. jannaschii hypothetical protein

MJ1073

YNL208w
weak similarity to Colletotrichum

gloeosporioides
nitrogen starvation-induced

glutamine rich protein

YNL213c
similarity to hypothetical protein Neurospora

crassa

YNL217w
weak similarity to E. coli bis(5′-nucleosyl)-

tetraphosphatase

YNL227c
similarity to dnaJ-like proteins

YNL230c
weak similarity to mammalian transcription
ELA1

elongation factor elongin A

YNL253w
similarity to hypothetical protein C. elegans

YNL255c
strong similarity to nucleic acid-binding
GIS2

proteins, similarity to Tetrahymena thermophila

cnjB prote

YNL260c
weak similarity to hypothetical protein

S. pombe

YNL275w
similarity to human band 3 anion transport

protein

YNL278w
similarity to YLR187w
CAF120

YNL279w
similarity to S. pombe coiled-coil protein of
PRM1

unknown function

YNL281w
strong similarity to YDR214w
HCH1

YNL294c
similarity to TRCDSEMBL:AF152926_1

pa1H Emericella nidulans

YNL308c
similarity to S. pombe and C. elegans
KRI1

hypothetical proteins

YNL311c
hypothetical protein

YNL313c
similarity to C. elegans hypothetical protein

YNL320w
strong similarity to S. pombe Bem46 protein

YNL321w
weak similarity to VCX1P

YNL323w
similarity to Ycx1p
LEM3

YNL334c
strong similarity to hypothetical proteins
SNO2

YFL060c and YMR095c

YNR018w
similarity to TRCDSEMBL:SPAC1565_1

hypothetical protein S. pombe

YNR021w
weak similarity to hypothetical protein

S. pombe

YNR039c
weak similarity to Anopheles mitochondrial
ZRG17

NADH dehydrogenase subunit 2

YNR047w
similarity to ser/thr protein kinases

YNR053c
strong similarity to human breast tumor

associated autoantigen

YNR054e
similarity to C. elegans hypothetical protein

CEESL47F

YNR065c
strong similarity to YJL222w, YIL173w and

PEP1P

YNR066c
strong similarity to PEP1P

Y0L010w
similarity to human RNA 3-terminal phosphate
RCL1

cyclase

Y0L027c
similarity to YPR125w

Y0L029c
hypothetical protein

Y0L034w
similarity to S. pombe RAD18 and rpgL29

genes and other members of the SMC

superfamily

YOL041c
weak similarity to M. sativa NUM1, hnRNP
NOP12

protein from C. tentans and D. melanogaster,

murine/bovine p

YOL045w
similarity to ser/thr protein kinase

YOL046c
questionable ORF

YOL054w
weak similarity to transcription factors

YOL063c
hypothetical protein

YOL075c
similarity to A. gambiae ATP-binding-cassette

protein

YOL077c
strong similarity to C. elegans K12H4.3 protein
BRX1

YOL078w
similarity to stress activated MAP kinase

interacting protein S. pombe

YOL082w
similarity to YOL083w
CVT19

YOL083w
similarity to YOL082w

YOL084w
similarity to A. thaliana hyp1 protein
PHM7

YOL087c
similarity to S. pombe hypothetical protein

YOL100w
similarity to ser/thr protein kinases
PKH2

YOL101c
similarity to YOL002c and YDR492w

YOL111c
weak similarity to human ubiquitin-like

protein GDX

YOL114c
similarity to human DS-1 protein

YOL117w
weak similarity to human sodium channel alpha

chain HBA

YOL128c
strong similarity to protein kinase MCK1P

YOL133w
similarity to Lotus RING-finger protein
HRT1

YOL138c
weak similarity to hypothetical trp-asp

repeats containing protein S. pombe

YOL146w
weak similarity to hypothetical protein

S. pombe

YOR054w
similarity to S. fumigata Asp FII

YOR001w
similarity to human nucleolar 100K
RRP6

polymyositis-scleroderma protein

YOR007c
similarity to protein phosphatases
SGT2

YOR009w
similarity to TIR1P and TIR2P
TIR4

YOR042w
weak similarity to YDR273w

YOR051c
weak similarity to nsyosin heavy chain

proteins

YOR054c
similarity to SIS2P protein and C. tropicalis

hal3 protein

YOR056c
weak similarity to human phosphorylation

regulatory protein HP-10

YOR066w
hypothetical protein

YOR073w
hypothetical protein

YOR080w
weak similarity to
DIA2

TRCDSEMBL:RNRNAHOP_1 Rattus

norvegicus
roRNA for Hsp70/Hsp90

organizing protein

YOR086c
weak similarity to synaptogamines

YOR093c
similarity to S. pombe hypothetical protein

SPAC22F3.04

YOR118w
similarity to PIR:T39884 hypothetical protein

SPBC21.02 S. pombe

YOR129c
weak similarity to hypothetical protein

SPBC776.06e S. pombe

YOR144c
weak similarity to human DNA-binding protein
EFD1

PO-GA and to bacterial H+-transporting

ATP synthases

YOR145c
strong similarity to hypothtical S. pombe

protein and to hypothetical C. elegans protein

YOR154w
similarity to hypothetical A. thaliana proteins

F19G10.15 and T19F06.21

YOR155c
similarity to 5′-flanking region of the Pichia

MOX gene

YOR164c
similarity to conserved hypothetical protein

S. pombe

YOR172w
similarity to finger protein YKL222c,

YOR162c and YLR266c

YOR173w
strong similarity to YLR270w

YOR177c
weak similarity to rat SCP1 protein

YOR186w
hypothetical protein

YOR191w
similarity to RAD5 protein
RIS1

YOR203w
questionable ORF

YOR206w
similarity to Brettanomyces RAD4 and to
(RAD4)

S. pombe
hypothetical protein

YOR214c
hypothetical protein

YOR215c
similarity to M. xanthus hypothetical protein

YOR220w
hypothetical protein

YOR226c
strong similarity to nitrogen fixation proteins
ISU2

YOR227w
similarity to microtubule-interacting protein

MHP1P

YOR256c
strong similarity to secretory protein SSP134P

YOR267c
similarity to ser/thr protein kinases

YOR269w
similarity to human LIS-1 protein
PAC1

YOR283w
weak similarity to phosphoglycerate mutases

YOR285w
similarity to D. melanogaster heat shock

protein 67B2

YOR296w
similarity to hypothetical S. pombe protein

YOR304c-a
similarity to mouse apolipoprotein A-IV

precursor

YOR304w
strong similarity to Drosophila ISW1 and
ISW2

human SNF2P homolog

YOR322c
similarity to hypothetical S. pombe protein

SPAC1F12.05

YOR324c
similarity to YAL028w

YOR339c
strong similarity to E2 ubiquitin-conjugating
UBC11

enzymes

YOR352w
hypothetical protein

YOR353c
weak similarity to adenylate cyclases

YOR356w
strong similarity to human electron transfer

flavoprotein-ubiquinone oxidoreductase

YOR367w
similarity to mammalian smooth muscle protein
SCP1

SM22 and chicken calponin alpha

YOR371c
similarity to YAL056w
GPE1

YOR378w
strong similarity to aminotriazole resistance

protein

YPL004c
strong similarity to YGR086c

YPL009c
similarity to M. jannaschii hypothetical

protein

YPL012w
hypothetical protein

YPL013c
strong similarity to N. crassa mitochondrial

ribosomal protein S24

YPL019c
strong similarity to YFL004w, similarity to
VTC3

YJL012c

YPL032c
strong similarityto PAM1P
SVL3

YPL034w
questionable ORF

YPL055c
hypothetical protein

YPL067c
hypothetical protein

YPL068c
hypothetical protein

YPL074w
similarity to VPS4P and YER047c
YTA6

YPL093w
similarity to M. jannaschii GTP-binding
NOG1

protein

YPL109c
similarity to aminoglycoside acetyltransferase

regulator from P. stuartii

YPL110c
similarity to C. elegans hypothetical protein,

weak similarity to PHO81P

YPL113c
similarity to glycerate dehydrogenases

YPL126w
weak similarity to fruit fly TFIID subunit p85
NAN1

YPL133c
weak similarity to transcription factors

YPL135w
strong similarity to nitrogen fixation protein
ISU1

(nifU)

YPL137c
similarity to microtubule-interacting protein

MHP1P and to hypothetical protein YOR227w

YPL138c
weak similarity to fruit fly polycomblike

nuclear protein

YPL146c
weak similarity to myosin heavy chain proteins

YPL150w
similarity to ser/thr protein kinases

YPL151c
strong similarity to A. thaliana PRL1 and
PRP46

PRL2 proteins

YPL156c
weak similarity to YDL010w
PRM4

YPL158c
weak similarity to human nucleolin

YPL166w
weak similarity to paramyosins

YPL168w
weak similarity to E. coli hfpB protein

YPL170w
similarity to C. elegans LIM homeohox protein

YPL176c
similarity to chinese hamster transferrin
SSP134

receptor protein

YPL181w
weak similarity to YKR029c

YPL184c
weak similarity to PUB1P

YPL191c
strong similarity to YGL082w

YPL206c
weak similarity to glycerophosphoryl diester

phosphodiesterases

YPL207w
similarity to hypthetical proteins from A.

fulgidus, M. thermoautotrophicum
and

M. jannaschii

YPL208w
similarity to YHL039w

YPL216w
similarity to YGL133w

YPL217c
similarity to human hypothetical protein
BMS1

KIAA0187

YPL222w
similarity to C. perfringens hypothetical protein

YPL226w
similarity to translation elongation factor eEF3
NEW1

YPL236c
similarity to PRK1P, and serine/threonine

protein kinase homolog from A. thaliana

YPL247c
similarity to human HAN11 protein and petunia

an11 protein

YPL249c
similarity to mouse Tbc1 protein

YPL253c
similarity to CIK1P
VIK1

YPL258c
similarity to B. subtilis transcriptional activator
THI21

tenA, and strong similarity to hypothetical

prote

YPL273w
strong similarity to YLL062c
SAM4

YPR003c
similarity to sulphate transporter proteins

YPR015c
similarity to transcription faetors

YPR021c
similarity to human citrate transporter protein

YPR022c
weak similarity to fruit fly dorsal protein

and SNF5P

YPR023c
similarity to human hypothetical protein
EAF3

YPR030w
similarity to YBL101c
CSR2

YPR037c
similarity to ERV1P and rat ALR protein
ERV2

YPR038w
questionable ORF

YPR040w
similarity to C. elegans C02C2.6 protein
SDF1

YPR049c
similarity to USO1P
CVT9

YPR078c
hypothetical protein

YPR085c
hypothetical protein

YPR090w
weak similarity to hypothetical protein

SPAC25B8.08 S. pombe

YPR091c
weak similarity to C. elegans LIM homeobox

protein

YPR093c
weak similarity to zinc-finger proteins

YPR105c
similarity to hypothetical protein SPCC338.13

S. pombe

YPR115w
similarity to probable transcription factor

ASK10P, and to YNL047c and YIL10Sc

YPR117w
similarity to YLR454w

YPR121w
similarity to B subtilis transcriptional activator
TH122

tenA

YPR130c
questionable ORF

YPR139c
weak similarity to nGAP H. sapiens

nGAP mRNA

YPR143w
hypothetical protein

YPR184w
similarity to human 4-alpha-glucanotransferase
GDB1

(EC 2 4.1.25)/amylo-1,6-glucosidase

(EC 3.2.1.33)

YPR188c
similarity to calmodulin and calinodulin-related
MLC2

proteins

Number	Date	Country
60323930	Sep 2001	US
60341213	Oct 2001	US
60345286	Jan 2002	US

Yeast proteome analysis

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

RELATED APPLICATIONS

Provisional Applications (3)