Patent Application
20100159486
Alzheimer's disease (AD) is a progressive degenerative disease of the brain primarily associated with aging. AD is one of several disorders that cause the gradual loss of brain cells and is one of and possibly the leading cause of dementia. Clinical presentation of AD is characterized by loss of memory, cognition, reasoning, judgment, and orientation. Mild cognitive impairment (MCI) is often the first identified stage of AD. As the disease progresses, motor, sensory, and linguistic abilities also are affected until there is global impairment of multiple cognitive functions. These cognitive losses occur gradually, but typically lead to severe impairment and eventual death in the range of three to twenty years.
An early diagnosis of AD has many advantages including additional time to make choices that maximize quality of life, less anxiety about unknown problems, a better chance of benefiting from treatment and more time to plan for the future. However, reliable noninvasive methods for diagnosing AD are not available.
Alzheimer's disease is characterized by two major pathologic observations in the brain: neurofibrillary tangles (NFT) and beta-amyloid plaques, comprised predominantly of an aggregate of fragments known as Aβ peptides. Individuals with AD exhibit characteristic beta-amyloid deposits in the brain (beta-amyloid plaques) and in cerebral blood vessels (beta-amyloid angiopathy) as well as neurofibrillary tangles. Neurofibrillary tangles occur not only in Alzheimer's disease but also in other dementia-inducing disorders. On autopsy, presently the only definitive method of diagnosing AD, large numbers of these lesions are generally found in areas of the human brain important for memory and cognition.
There is an urgent clinical need to develop diagnostic markers that can detect early stage AD, particularly at the stage of MCI. While advances have been made in imaging beta-amyloid, (Lopresti et al. J. Nucl. Med. (2005) 46:1959-1972), no serum biomarkers for AD are clinically available. To date there are no validated biomarkers for confirming the diagnosis of a major neurodegenerative disorder or to monitor progression (Castano et al. Neurol. Res. (2006) 28:1155-163).
Despite the enthusiasm for the use of proteomic technology to discover blood markers of AD, and decades of effort, progress towards identifying useful markers has been slow, possibly because putative high specificity AD markers are assumed to be in very low abundance because they are shed from small volumes of diseased tissue and are expected to be rapidly cleared and metabolized. In addition, researchers have avoided studying blood because the blood proteome is dominated by, and complicated by, resident proteins such as albumin that can exist at a concentration many millions of times greater than the target low abundance biomarker. For this reason, researchers have focused on cerebrospinal fluid (CSF) as the target fluid for AD biomarkers (see Zhang et al., J. Alzheimer's Disease (2005) 8:377-3386). The CSF approach, however, has limited clinical application to routine screening. Moreover, the blood brain vascular circulation perfuses AD lesions with a higher efficiency, particularly in the case for amyloid angiopathy.
In one aspect, methods are provided for diagnosing a neurological condition in a patient comprising obtaining a biological sample from the patient and evaluating the sample for the abundance of at least one biomarker selected from the group consisting of a peptide having the amino acid sequence of SEQ ID NOs:1-440, wherein the abundance of said at least one biomarker is indicative of a neurological condition. In one embodiment, the abundance of the biomarker is greater than that of a control sample. In another embodiment, the abundance of the biomarker is less than that of a control sample.
The method also can comprise, prior to the evaluation step, harvesting low molecular weight peptides from said sample to generate at least one fraction comprising said peptides. The biomarker can be a low molecular weight protein complexed with a carrier protein. In a further embodiment, the low molecular weight protein is further purified from said carrier protein. In another embodiment, the low molecular weight protein is digested and optionally sequenced. In one embodiment, the biological sample is blood, serum or plasma. In another embodiment, the evaluation step comprises an assay selected from the group consisting of mass spectrometry, such as tandem mass spectrotrometry (MS MS), immunoassay, such as enzyme-linked immunosorbent assay (ELISA), immuno-mass spectrometry and suspension bead array. The method also can comprise obtaining a neuroimage of the brain microvasculopathy, which can be optionally obtained using susceptibility weighted imaging, perfusion weighted imaging and magnetic resonance spectroscopy.
The neurological condition can be Alzheimer's disease (AD), mild cognitive impairment (MCI), stable mild cognitive impairment (stable MCI), progressive mild cognitive impairment (PMCI), vascular dementia (VD), angiopathy black holes, cerebral amyloid angiopathy (CAA) and brain microhemorrages. In one embodiment, methods are provided for diagnosing Alzheimer's disease in a patient comprising obtaining a biological sample from said patient, and evaluating said sample for the abundance of at least one biomarker selected from the group consisting of a peptide having the amino acid sequence of SEQ ID NOs:1, 3-13, 15, 16, 21, 22, 24-28, 31-33, 37-44, 56-59, 66-68, 93-101, 111-128, 143-153, 156-1170, 172-183, 263-279, 310-335, 348, 355-359, 362, 363, 365, 372, 373, 376-402, 406-426 and 436-44, wherein the abundance of said at least one biomarker is indicative of Alzheimer's disease. In another aspect, the biomarker is a peptide associated with a metabolic pathway or cellular process. In others aspects, the biomarker is a peptide associated with inflammation, estrogen activity, pigment epithelium-derived factor (PEDF), vitamin D metabolism and bone mineralization, coagulation and platelet activity, the complement cascade, acyl-peptide hydrolase (APH) activity, vitamin A and thyroxine, phospholipase activity, globin activity, glycosylation or is glycosylated, protease inhibition, keratins and related proteins, heme degradation, pyruvate metabolism, calcium related proteins, defensin, gelsolin, vitronectin, profilin, thrombospondin, peroxiredoxin, alcohol dehydrogenase, apolipoproteins, iron and copper metabolism, or NMDA receptor-related proteins.
In another aspect, methods are provided for diagnosing mild cognitive impairment in a patient comprising obtaining a biological sample from the patient and evaluating the sample for the abundance of at least one biomarker selected from the group consisting of a peptide having the amino acid sequence of SEQ ID NOs: 2, 4, 14, 17, 23, 29, 34, 45-55, 60-65, 69-92, 102-110, 129-142, 154, 155, 171, 184-191, 193-226, 248-279, 281-320, 333, 336-347, 349-354, 360, 361, 364, 366-371, 374, 375, 403-405 and 427-435, wherein the abundance of said at least one biomarker is indicative of mild cognitive impairment.
In yet another aspect, methods are provided for diagnosing brain microhemorrhages in a patient comprising obtaining a biological sample from the patient and evaluating the sample for the abundance of at least one biomarker selected from the group consisting of a peptide having the amino acid sequence of SEQ ID NOs:441-452, wherein the abundance of said at least one biomarker is indicative of brain microhemorrhages.
In some embodiments, the inventive methods comprise, prior to the evaluation step, harvesting low molecular weight peptides from the biological sample to generate at least one fraction comprising the peptides. The size of the low molecular weight peptides can be, for example, less than 50 KDa, less than 25 KDa, or less than 15 KDa. The methods also can comprise digesting the low molecular weight peptides. Such digestion can be accomplished using enzymatic or chemical means. In one example, trypsin can be used to digest the peptides.
In other aspects, antibodies are provided that are specific for biomarkers for a neurological condition, as well as kits for detecting a neurological condition in a patient, comprising at least one such antibody. The antibody can be, for example, a monoclonal or polyclonal antibody, and also be a chimeric, humanized or human antibody.
Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples where it will be obviously useful to those skilled in the prior art.
Low molecular weight (LMW) peptides have been discovered from the repertoire of proteins bound to carrier proteins such as albumin that are indicative of a neurological condition. Evaluating patient samples for the presence of such LMW peptides is an effective means of detecting a neurological condition and monitoring the progression of the disease, for example during treatment. The LMW peptides are particularly useful in detecting a neurological condition during its early stages. The LMW peptides are particularly useful for detecting AD, MCI and brain microhemorrhages.
The LMW peptides, which are biomarkers, can be detected using a variety of methods known in the art. For example, antibodies can be utilized in immunoassays to detect the presence of a biomarker. Exemplary immunoassays include, e.g., ELISA, radioimmunoassay, immunofluorescent assay, “sandwich” immunoassay, western blot, immunoprecipitation assay and immunoelectrophoresis assays. In other aspects, microbeads, arrays, microarrays, etc. can be used in detecting the LMW peptides. Exemplary assays include, but are not limited to, a suspension bead assay (Schwenk et al., “Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics,” Mol. Cell Proteomics, 6(1): 125-132 (2007)), an antibody microarray (Borrebaeck et al., “High-throughput proteomics using antibody microarrays: an update,” Expert Rev. Mol. Diagn. 7(5): 673-686 (2007)), an aptamer array (Walter et al., “High-throughput protein arrays: prospects for molecular diagnostics,” Trends Mol. Med. 8(6): 250-253 (2002)), an affybody array (Renberg et al., “Affibody molecules in protein capture microarrays: evaluation of multidomain ligands and different detection formats,” J. Proteome Res. 6(1): 171-179 (2007)), and a reverse phase array (VanMeter et al., “Reverse-phase protein microarrays: application to biomarker discovery and translational medicine,” Expert Rev. Mol. Diagn. 7(5): 625-633 (2007)). All of these publications are incorporated herein by reference.
In another example, the inventive biomarkers can be detected using mass spectrometry (MS). One example of this approach is tandem mass spectrometry (MS/MS), which involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. Most such assays use electrospray ionization followed by two stages of mass selection: a first stage (MS1) selecting the mass of the intact analyte (parent ion) and, after fragmentation of the parent by collision with gas atoms, a second stage (MS2) selecting a specific fragment of the parent, collectively generating a selected reaction monitoring assay. In one embodiment, collision-induced dissociation is used to generate a set of fragments from a specific peptide ion. The fragmentation process primarily gives rise to cleavage products that break along peptide bonds. Because of the simplicity in fragmentation, the observed fragment masses can be compared to a database of predicted masses for known peptide sequences. A number of different algorithmic approaches have been described to identify peptides and proteins from tandem mass spectrometry (MS/MS) data, including peptide fragment fingerprinting (SEQUEST, MASCOT, OMSSA and X!Tandem), peptide de novo sequencing (PEAKS, LuteFisk and Sherenga) and sequence tag based searching (SPIDER, GutenTAG).
Likewise, multiple reaction monitoring (MRM) can be used to identify the inventive biomarkers in patient samples. This technique applies the MS/MS approach to, for example, tryptic digests of the input sample, followed by selected ion partitioning and sampling using MS to make the analyte selection more objective and discrete by following the exact m/z ion of the tryptic fragment that represents the analyte. Such an approach can be performed in multiplex so that multiple ions can be measured at once, providing an antibody-free method for analyte measurement. See, e.g. Andersen et al., Molecular & Cellular Proteomics, 5.4: 573-588 (2006); Whiteaker et al., J. Proteome Res. 6(10): 3962-75 (2007). Both publications are incorporated herein by reference.
In another example, the inventive biomarkers can be detected using nanoflow reverse-phase liquid chromatography-tandem mass spectrometry. See, e.g., Domon B, Aebersold R. Science, 312(5771):212-7(2006), which is incorporated herein by reference. Using this approach, practitioners obtain peptide fragments, usually by trypsin digest, and generate mass spectrograms of the fragments, which are then compared to a database, such as SEQUEST, for protein identification.
In another aspect, the inventive biomarkers can be detected using immuno-mass spectrometry. See, e.g., Liotta L et al. J Clin Invest.,116(1):26-30 (2006), Nedelkov, Expert Rev. Proteomics, 3(6): 631-640 (2006), which are incorporated herein by reference. Immuno-mass spectrometry provides a means for rapidly determining the exact size and identity of a peptide biomarker isoform present within a patient sample. When developed as a high throughput diagnostic assay, a drop of patient's blood, serum or plasma can be applied to a high density matrix of microcolumns or microwells filled with a composite substratum containing immobilized polyclonal antibodies, directed against the peptide marker. All isoforms of the peptide that contain the epitope are captured. The captured population of analytes including the analyte fragments are eluted and analyzed directly by a mass spectrometer such as MALDI-TOF MS. The presence of the specific peptide biomarker at its exact mass/charge (m/z) location can be used as a diagnostic test result. The analysis can be performed rapidly by simple software that determines if a series of ion peaks are present at defined m/z locations.
In yet another example, the inventive biomarkers can be detected using standard immunoassay-based approaches whereby fragment specific antibodies are used to measure and record the presence of the diagnostic fragments. See, e.g., Naya et al. “Evaluation of precursor prostate-specific antigen isoform ratios in the detection of prostate cancer.” Urol Oncol. 23(1):16-21 (2005). Moreover, additional immunoassays are well known to one skilled in the field, such as ELISA (Maeda et al., “Blood tests for asbestos-related mesothelioma,” Oncology 71: 26-31 (2006)), microfluidic ELISA (Lee et al., “Microfluidic enzyme-linked immunosorbent assay technology,” Adv. Clin. Chem. 42: 255-259 (2006)), nanocantilever immunoassay (Kurosawa et al., “Quartz crystal microbalance immunosensors for environmental monitoring,” Biosens Bioelectron, 22(4): 473-481 (2006)), and plasmon resonance immunoassay (Nedelkov, “Development of surface Plasmon resonance mass spectrometry array platform,” Anal. Chem. 79(15): 5987-5990 (2007)). All publications are incorporated herein by reference.
In a further example, the inventive biomarkers can be detected using electrochemical approaches. See, e.g., Lin et al., Anal. Sci. 23(9): 1059-1063 (2007)).
In one embodiment, the LMW peptides are harvested from a biological sample prior to the evaluation step. For example, 100 μl of serum can be mixed with 2×SDS-PAGE Laemmli Buffer (containing 200 mM DTT), boiled for 10 minutes, and loaded on Prep Cell (Model 491 Prep Cell, Bio-Rad Laboratories, Calif.) comprising a 5 cm length 10% acrylamide gel. Electrophoresis is performed under a constant voltage of 250V. Immediately after the bromophenol blue indicator dye is eluted from the system, LMW peptides and proteins migrate out of the gel and are trapped in a dialysis membrane in the elution chamber. These molecules can be eluted at a flow rate of 400 ml/min by a buffer with the same composition of the Tris-Glycine running buffer and collected for 10 minutes in one fraction.
Alternatively, LMW peptides can be harvested from a sample using a capture-particle that comprises a molecular sieve portion and an analyte binding portion as described in U.S. patent application Ser. No. 11/527,727, filed Sep. 27, 2006, which is incorporated herein by reference. Briefly, either the molecular sieve portion or the analyte binding portion or both comprise a cross-linked region having modified porosity, or pore dimensions sufficient to exclude high molecular weight molecules.
In another embodiment, the LMW peptides are digested prior to detection, so as to reduce the size of the peptides. Such digestion can be carried out using standard methods well known in the field. Exemplary treatments, include but are not limited to, enzymatic and chemical treatments. Such treatments can yield partial as well as complete digestions. One example of an enzymatic treatment is a trypsin digestion.
The inventive biomarkers are particularly useful in detecting a neurological condition during its early stages, such as while the condition is still associated with MCI or PMCI or for detecting brain vasculopathy, such as brain microhemorrhages. For clarification, mild cognitive impairment (MCI) cases fulfill the Mayo Clinic criteria for classification as MCI-multiple domain impairment (MCI-MCDI) with the following characteristics: i) A memory complaint confirmed by either corrected Logical Memory testing or reports of the informant and a CDR=0.5. ii) Normal activities of daily living. iii) Normal general cognitive function. iv) Abnormal memory for age as measured by standard scores and education. v) A global CDR of 0.5 and no dementia. vi) No history of significant vascular problems, insulin-requiring diabetes, or uncontrolled hypertension. Meanwhile, stable mild cognitive impairment (stable MCI) is based on a Sum of boxes=0.5-3.5 on several evaluations, CDR logical memory impairment with logical memory impairment on at least one evaluation, neuropsychological testing in MCI range inconsistently and clinical judgment. Progressive mild cognitive impairment (PMCI) denotes patients with a Sum of Boxes≧3.5 on two occasions, neuropsychological tests congruent with CDR, a Logical Memory raw score low to zero and clinical judgment.
The abundance of the biomarker can be measured by detecting the biomarker as described above and comparing the amount of the biomarker to a control. The abundance of the biomarker is an indicator of the neurological condition. If the biomarker is “less abundant” in the control, then the biomarker is present in the tested sample in a significantly less amount than in the control sample. If the biomarker is “more abundant” than the control, then the biomarker is present in the tested sample in a significantly greater amount than in the control sample. For instance, the difference may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, or greater. The control can be a sample or its equivalent from a normal patient or from a patient in a known disease state. For instance, the control can be from a patient with AD, MCI or brain microhemorrhages. The control can also be a standard or known amount of a reference peptide.
The neurological condition being detected can be, for example, Alzheimer's disease (AD), mild cognitive impairment (MCI), stable mild cognitive impairment (stable MCI), progressive mild cognitive impairment (PMCI), vascular dementia (VD), angiopathy black holes, cerebral amyloid angiopathy (CAA) and brain microhemorrhages. Unless otherwise indicated, the conditions and activities noted herein refer to the commonly accepted definitions thereof. For instance, as described in more detail in the Examples, cognitive impairment is defined according to the Mayo Clinic criteria.
In another embodiment, the biomarker is a peptide associated with a metabolic pathway or cellular process. In further embodiments, the biomarker is a peptide associated with inflammation, estrogen activity, pigment epithelium-derived factor (PEDF)vitamin D metabolism and bone mineralization, coagulation and platelet activity, the complement cascade, acyl-peptide hydrolase (APH) activity, vitamin A and thyroxine, phospholipase activity, globin activity, glycosylation or is glycosylated, protease inhibition, keratins and related proteins, heme degradation, pyruvate metabolism, calcium related proteins, defensin, gelsolin, vitronectin, profilin, thrombospondin, peroxiredoxin, alcohol dehydrogenase, apolipoproteins, iron and copper metabolism, or NMDA receptor-related proteins.
In one aspect, more than one biomarker can be evaluated simultaneously. For example, at least two, at least five, at least 10, at least 20, at least 30, at least 50, at least 75, at least 100 biomarkers are evaluated in the methods. Analyzing more than one biomarker can increase accuracy of the diagnosis.
The present methods can be combined with neuroimaging techniques for the detection of neuropathy and brain microvasculopathy associated with a neurological condition. For example, neuroimaging can be used to detect brain microhemorrages associated with cognitive impairment. Using magnetic resonance imaging, focal signal intensity losses secondary to iron-containing hemosiderin residuals can be detected. These spots on the MR image have been termed “signal voids,” “susceptibility artifacts,” “black holes,” “dots,” “microbleeds,” “old microbleeds” (OMBs), “multifocal signal loss lesions” or “microhemorrhages” (MH). Generically, these spots are called small hypointensities (SH) and are associated with AD and MCI (Cordonnier et al. Neurology (2006) 66:1356-1360; Werring et al. Brain (2004) 127:2265-2275). Suitable MR imaging techniques include gradient refocused echo T2* (GRE-T2) and susceptibility weighted imaging (SWI).
Neuroimaging methods that detect metabolic changes in the brain also can be used in conjunction with the present biomarkers. MR spectroscopy that detects, for instance, differences in neurotransmitters, such as glutamine, glutamate and gamma-aminobutryic acid (GABA), can be used to analyze changes in these systems associated with a neurological condition. These metabolic changes can be correlated with cognitive decline and biomarker abundance.
Antibodies specific for the inventive biomarkers can be produced readily using well known methods in the art. (See, J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, pp. 18.7-18.18, 1989) For example, the inventive biomarkers can be prepared readily using an automated peptide synthesizer. Next, injection of an immunogen, such as (peptide)n-KLH (n=1-30) in complete Freund's adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund's adjuvant into immunocompetent animals, is followed three days after an i.v. boost of antigen, by spleen cell harvesting. Harvested spleen cells are then fused with Sp2/0-Ag14 myeloma cells and culture supernatants of the resulting clones analyzed for anti-peptide reactivity using a direct-binding ELISA. Fine specificity of generated antibodies can be detected by using peptide fragments of the original immunogen.
In certain embodiments, one or more antibodies directed to the inventive biomarkers is provided in a kit, for use in a diagnostic method. Such kits also can comprise reagents, instructions and other products for performing the diagnostic method.
In other aspects, the biomarkers and antibodies of the present invention are useful for discovering novel aspects of neurological conditions, such as those described herein.
The following examples are illustrative only, and should not be construed as limiting. Also, each reference disclosed herein, and throughout the specification, is incorporated by reference in its entirety.
A community-based cohort of 103 participants (75 MCI and 28 cognitively normal subjects) was recruited for the study. Of the original 75 MCI subjects, 20 have been censored from the study for various reasons not related to dementia, leaving 55 which are currently being followed. Seventeen of these have become demented over a 0.5 to 4.1-year observation period (15% annual conversion rate) based upon on the Clinical Dementia Rating (CDR) Sum of Boxes score≧3.5 as documented by NINCDS-ADRDA criteria.(Schafer et al. Alzheimer Dis Assoc Disord.(2004) 18:219-222; McKhann et al. Neurology.(1984) 34:939-944) Four of 28 cognitively normal subjects have progressed to the MCI category with significant SH detected by SWI in two. Two MCI cases are on the verge of dementia at present, one with significant SH. SWI brain imaging has demonstrated increasing and “significant” numbers (n≧5) of SH in 7 of the 17 demented and progressively cognitively impaired subjects. This progressive increase in SH in a lobar, posteriorly situated cortical-subcortical pattern fits the diagnostic pattern for “probable CAA.”(Knudsen et. al. Neurology. (2001) 56:537-539) This observation is the first prospective evidence for a subset of sporadic late-onset dementia correlating temporally with increasing SH in a pattern typical for CAA.
After screening 1348 community based individuals at publicized memory clinics, 28 elderly “controls” and 75 subjects with MCI qualified for the study using inclusion and exclusion criteria defined by the Mayo Clinic Group (Petersen R C, et al. Arch Neurol. March (1999) 56:303-308.) Subjects have been continuously evaluated with serial cognitive (bi-yearly) and radiologic (yearly) procedures over 4.1 years (range 0.5 to 4.10 years, average total follow-up time 2.3±1.2 years, total person years of follow-up 241.7 years). All subjects gave informed consent and all studies were approved by the Loma Linda University Institutional Review Board. Complete medication, medical and smoking histories were obtained on all subjects, and thyroid function, serum B12 levels, and ApoE genotype were defined in all subjects.
Normal Subjects: (n=28)
All “control subjects” were without objective or subjective memory deficits and within normal limits on neuropsychological testing (Global CDR of 0, CDR memory component of 0 and a sum of CDR boxes of 1 or less at baseline). The sum of CDR boxes is used as a measure of cognitive performance.(107)
MCI Subjects: (n=75)
All MCI cases fulfilled the Mayo Clinic criteria for classification as MCI-multiple domain impairment (MCI-MCDI) with the following characteristics: i) a memory complaint confirmed by either corrected Logical Memory testing or reports of the informant and a CDR=0.5; ii) normal activities of daily living. iii) normal general cognitive function; iv) abnormal memory for age as measured by standard scores and education; v) a global CDR of 0.5 and no dementia; and vi) no history of significant vascular problems, insulin-requiring diabetes, or uncontrolled hypertension. Twenty MCI subjects have now been censored for varying reasons: cancer 2, co-morbidity 1, claustrophobia 2, loss of care support/moved 9, lost interest 5 and pacemaker 1.
All cognitive assessments were conducted within 4 weeks of the MR evaluation by the same team of neuropsychologists with re-evaluations at approximately 6 month intervals. A total of 476 cognitive tests have been performed with some subjects having as many as 9 evaluations. The battery of cognitive tests included a videotaped CDR plus the following: Logical Memory I, II, North American Adult Reading Test, Word Fluency:Phonetic and Semantic, Wisconsin Card Sorting Test, Trail Making Test A&B, Boston Naming Test, Draw-A-Clock, Depression Features Battery Version II, and Geriatric Depression Scale.
Results of radiologic and cognitive assessments were reviewed bimonthly. On the rare occasion if cognitive testing and neurologic examination indicates development of a disorder other than AD, e.g. frontotemporal dementia, progressive supranuclear palsy, primary progressive aphasia, the subject was removed from the study. Results of the neuropsychological testing were noted as abnormal if below>1.5 standard deviation (SD) on normative data based on age and education. The diagnosis of dementia is based on a clinical judgment (consensus conference), NINCDS-ADRDA criteria, and a Sum of Boxes (SOB) on the CDR≧3.5.(107)
The cognitive course of the cohorts has been carefully monitored over the past 4.1 years and a five stage classification has emerged (Table 5). This classification is the matrix on which the MR and proteomic findings are co-analyzed. Special attention has been given to the MCI and control cases that under observation have proceeded to cognitive loss (MCI), “dementia,” or “progressed MCI.”
The above scoring was derived after examination of results of multiple NP evaluations. Subjects with only one evaluation at baseline are classed as Normal or MCI.
Clear fluctuations in cognitive performance were found in both the Unstable Normal and Unstable MCI cohorts. The unstable MCI cohort has a cognitive status on occasion of dementia (CDR=3.5) but can improve to 3.0 with medication. A complete medication history has been obtained on all cohorts.
Table 7 gives the current NP status of the cohorts using the five stage classification as derived from entrance classification (normal or MCI). Note progressive movement of normal to MCI and 10 MCI cases moving to U-Normal and Normal, 25 of the MCI cases have moved to U-MCI (8) and PMCI (17). The human experiment was designed to determine MR and proteomic changes during dementia development.
Low molecular weight protein harvesting by PrepCell
100 μl of serum was mixed with SDS-PAGE loading buffer, boiled for 10 minutes, and loaded to PrepCell (Bio-Rad, CA). After 2 hours of electrophoresis, low molecular weight proteins migrated out of the gel and were eluted to collection tubes.
Nanoflow reversed-phase liquid chromatography-tandem MS (nanoRPLC-MS/MS)
Eluted proteins from PrepCell were further passed through detergent clean-up micro kit ProteoSpin (Norgen, Canada) to remove the SDS in the elution buffer that could interfere with mass spectrometry analysis. The cleaned proteins were reduced by 10 mM DTT, alkylated by 50 mM iodoacetamide, and digested by trypsin (from Promega) at 37° C. overnight. Tryptic peptides were further purified by Sep-Pak cartridges (Waters, Mass.) and analyzed by reversed-phase liquid chromatography nanospray tandem mass spectrometry using a linear ion-trap mass spectrometer (LTQ, ThermoElectron, San Jose, Calif.). Separation column was slurry-packed in-house with 5 μm, 200 Å pore size C18 resin (Michrom BioResources, CA) in 100 μm i.d.×10 cm long fused silica capillary (Polymicro Technologies, Phoenix, Ariz.) with a laser-pulled tip. After sample injection, the column was washed for 5 minutes with mobile phase A (0.4% acetic acid) and peptides were eluted using a linear gradient of 0% mobile phase B (0.4% acetic acid, 80% acetonitrile) to 50% mobile phase B in 30 minutes at 250 nanoliter/min, then to 100% B in an additional 5 minutes. The LTQ mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS scans where the five most abundant molecular ions were dynamically selected for collision-induced dissociation (CID) using a normalized collision energy of 35%.
The ETD method with Thermo LTQ instrument also can be used. The ETD method (Syka et al. Proc. Natl. Acad. Sci. U.S.A. (2004) 101:9528-9533) accomplishes peptide fragmentation in the MS-MS analysis by electron transfer, in contrast to the traditional collision-induced dissociation (CID). ETD has been demonstrated to be more powerful than CID in providing more easily interpretable MS-MS sequence data from larger, higher-charge state peptides (including intact small proteins), as well as those with post-translational modifications (PTMs). (Coon et al. Proc. Natl. Acad. Sci. U.S.A. (2005) 102:9463-9468). The novel combination of CID and ETD analysis can enhance peptide identification productivity.
Fractionating LMW Proteins
In the first serum proteomic study, A, 100-μL aliquots of whole serum samples were prepared for high performance liquid chromatography/mass spectrometry (LC-MS) analysis by reduction and alkylation (DTT, iodoacetamide) followed by digestion of the proteins followed by LTQ mass spectroscopy. For subsequent studies, B and C a proteome subset consisting of low molecular weight (LMW) proteins was prepared from each serum sample to reduce the complexity of the protein mixture. The resulting LMW proteins were fractionated by SDS-PAGE and proteins were visualized by Coomassie staining.
For study B, the samples consisted of pooled serum samples from 14-15 subjects (control, MCI and PMCI). With improved LMW isolation, serum proteins with molecular weights 25 kDa were collected and fractionated by SDS-PAGE.
For study C serum samples from 5 individuals who had progressed from control to MCI (1 sample) and from MCI to PMCI were prepared to yield LMW proteins and the LC-MS analyses performed using a Thermo hybrid LTQ-Orbitrap mass spectrometer. This represents the state of the art in the MS technology and provides several advantages compared with the LTQ, such as superior high mass resolution and mass accuracy in the spectra acquired of the precursor peptide molecular ions.
MS-MS spectra were searched against a public human protein database (NCBI) using the SEQUEST search algorithm to obtain matches. Results in study A only identified abundant serum proteins. The results led to a focus on low molecular weight (LMW) serum proteins (study B). The threshold of 50 kDa was insufficient to reduce the complexity of proteins, and TCA protein precipitation resulted in unacceptable protein loss. As a result, a high-quality analysis of study B was conducted using pooled samples of a relatively large number (14) of individual subject serum samples per group. This study compared LMW proteins identified in control vs. MCI vs. PMCI sample/subject groups. This qualitative analysis identified candidate biomarkers (differentially abundant proteins). The objective of study C was to identify LMW serum proteins with differential abundances that correlated with progression from MCI to PMCI (4 individuals; 4 sample pairs) and control to MCI (1 individual; 1 pair of samples) diagnoses. These 10 sample analyses yielded identification of more than 500 proteins. No major differences in apoE genotype between subjects are found in the subject cohorts.
Determination of candidate biomarker proteins was achieved by comparing the number of tandem mass spectra (MS2 scans) that were matched to peptide sequences corresponding to the source proteins in the database against which the data were searched. A higher abundance protein relative to a lower abundance one will yield a greater number of, and more abundant, peptides from the enzyme digest, and these peptides often will result in more matched MS2 spectra. In this way, the number of MS2 spectra, termed “spectral count”, is an approximate measure of the relative abundance of proteins in a mixture (Analytical Chemistry, 76(14), 4193-4201 (2004)). The evaluation of candidate differentially abundant proteins focuses on proteins that yielded a 50% or greater spectral count difference in one sample set versus the other.
The results of the studies are shown in Tables 8-10.
SH are counted independently at two sites (Detroit MRI Institute for Biomedical Research (DMRI) and Loma Linda University (LLU)) but currently primarily at LLU by raters who are integral to the project using an identical protocol blinded to clinical status. SWI filtered phase images were reviewed for the presence of SH one 2 mm slice at a time. All magnitude images, high pass (HP) filtered phase images and contrast enhanced SWI magnitude images were used in the data review process. Images were placed side by side for identifying SH and HP filtered phase images are used to mark them with review above and below to check for vascular connections. One slice may contain more than one SH as in FIG. 2., then every SH was highlighted with a different colored boundary. Any slice that showed a SH appearing in a previous slice was not recounted. SH are assigned a slice and serial number, size (1-3, 3-5, >5 mm O.D.) and anatomical location. Differentiating microaneurysms with blood in and/or around vessel walls was uncertain since blood collecting in a microaneurysm produces a significant signal void. Subarachnoid and sulcal vascular voids, symmetrical focal basal ganglia signal losses were not counted.
The biomarkers identified as associated with brain microhemorrages are presented in Table 11.
The inventive biomarkers can be evaluated further using a variety of methods. In addition to traditional biological validation assays, mass spectrometric methods can be used. One method of validation is Western assays of serum samples using commercially available antibodies specific for the candidate proteins. If antibodies are not available commercially, they can be produced readily using methods well know in the art and disclosed herein.
In addition, triple quadruple mass spectrometry (TQMS) technology can be used to further evaluate the biomarkers. The technique employs multiple reaction monitoring (MRM), which consists of (1) detection and selection of molecular ions with the first quadruple, (2) fragmentation of these ions in the second quadruple, and (3) detection of a small number of known fragment ions in the third quadruple. The analysis yields an analyte's molecular weight and the relative abundances of fragment ions that are characteristic of analyte structure and chromatographic elution time (LC/MS). Modern TQMS instruments provide advanced MRM performance with higher resolution and accuracy mass measurement, fast electronics for switching between a large number of selected analyte and fragmentation masses monitored, and ease of use. Inherent advantages of LC/TQMS include high detection sensitivity, large dynamic range of detection response, and the ability to incorporate stable isotope labeled synthetic analogs of the targeted analytes, which allows superior quantitative analytical performance.(Anderson, Mol. Cell. Proteomics (2006) 5:573-588; Frewen et al. Anal. Chem. (2006) 78:5678-5684)
Such studies can be augmented with spiked internal standards, as in the discovery phase, and with isotopically-labeled synthetic analogs of the biomarkers. In addition, an autosampler and other methods can be used to enhance throughput (e.g., plate-based sample peptide enrichment and cleanup prior to LC/MS).
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
Homo sapiens]
P13645
(P13645) Keratin, type I cytoskeletal 10 (Cytokeratin-10) (CK-
59502.3
0
57
4
100%
100%
87%
10) (Keratin-10) (K10)
P35908
(P35908) Keratin, type II cytoskeletal 2 epidermal
65848.4
0
16
4
100%
100%
60%
(Cytokeratin-2e) (K2e) (CK 2e)
P02671
(P02671)
Fibrinogen alpha chain precursor [Contains:
94955.4
1
5
7
75%
67%
17%
Fibrinopeptide A]
Q6PYX1
(Q6PYX1) Hepatitis B virus receptor binding protein
38143
1
8
5
67%
78%
23%
(Fragment)
O75179
(O75179) KIAA0697 protein (Fragment)
263225.4
1
2
5
67%
33%
43%
P55056
(P55056) Apolipoprotein C-IV precursor (Apo-CIV) (ApoC-IV)
14535.5
2
2
7
56%
0%
56%
more abundant in mild AD
P16591
FER_HUMAN
1.25E−04
94563.8
0.00
0.00
0.00
0.40
0.67
100%
0%
100%
100%
25%
100%
100%
(P16591) Proto-
oncogene
tyrosine-protein
kinase FER (EC
2.7.1.112) (p94-
FER) (c-FER)
P22792
CPN2_HUMAN
2.00E−07
60576.3
0.00
0.00
0.00
0.60
0.58
100%
0%
100%
100%
1%
100%
100%
(P22792)
Carboxypeptidase
N subunit 2
precursor
(Carboxypeptidase
N polypeptide
2) (Carb
Q6ZVQ3
Q6ZVQ3
9.19E−04
17595.2
0.00
0.00
0.00
0.80
0.50
100%
0%
100%
100%
23%
100%
100%
(Q6ZVQ3)
Hypothetical
protein FLJ42220
Q9H2I2
Q9H2I2 (Q9H2I2)
2.53E−04
31787.4
0.00
0.00
0.00
0.20
0.33
100%
0%
100%
100%
25%
100%
100%
HNRBF-2
P13667
PDIA4_HUMAN
2.62E−04
72887.1
0.00
0.00
0.00
0.20
0.33
100%
0%
100%
100%
25%
100%
100%
(P13667) Protein
disulfide-
isomerase A4
precursor (EC
5.3.4.1) (Protein
ERp-72) (ERp72)
O60602
TLR5_HUMAN
2.76E−04
97663.4
0.00
0.00
0.00
0.00
0.33
100%
0%
0%
0%
100%
100%
100%
(O60602) Toll-
like receptor 5
precursor
(Toll/interleukin-
1 recepto
O95793
STAU_HUMAN
2.83E−04
63227.9
0.00
0.00
0.00
0.40
0.33
100%
0%
100%
100%
9%
100%
100%
(O95793)
Double-
stranded
RNA-binding
protein
Staufen
homolog
Q7Z3Z2
CA036_HUMAN
8.02E−05
22689.6
0.00
0.00
0.00
0.20
0.33
100%
0%
100%
100%
25%
100%
100%
(Q7Z3Z2)
Protein
C1orf36
Q05513
KPCZ_HUMAN
1.14E−04
67616.8
0.00
0.00
0.00
0.40
0.33
100%
0%
100%
100%
9%
100%
100%
(Q05513)
Protein kinase
C, zeta type
(EC 2.7.1.37)
(nPKC-zeta)
Q4V312
Q4V312
5.36E−05
46871.6
0.00
0.00
0.00
0.60
0.33
100%
0%
100%
100%
29%
100%
100%
(Q4V312)
Colony
stimulating
factor 2
receptor,
alpha, low-
affinity
(Granulocyte-
macrophage)
Q93088
BHMT_HUMAN
6.52E−04
44941.8
0.00
0.25
0.14
0.60
0.75
68%
100%
100%
41%
11%
50%
100%
(Q93088)
Betaine--
homocysteine
S-
methyltransferase
(EC
2.1.1.5)
Q8N7W7
Q8N7W7
5.41E−04
66086.9
0.00
0.25
0.14
0.40
0.58
61%
100%
100%
23%
19%
40%
100%
(Q8N7W7)
Hypothetical
protein
FLJ40259
P35542
SAA4_HUMAN
9.09E−04
14797.3
0.00
0.25
0.14
0.00
0.58
61%
100%
0%
100%
100%
40%
100%
(P35542)
Serum
amyloid A-4
protein
precursor
(Constitutively
expres
Q9BXB9
Q9BXB9
2.79E−04
46480
0.00
0.25
0.14
0.60
0.50
56%
100%
100%
41%
9%
33%
100%
(Q9BXB9) LIM
mineralization
protein 2
more abundant in mild AD
P02654
(P02654) Apolipoprotein C-I precursor (Apo-CI) (ApoC-I)
9314.4
6
2
9
20%
50%
64%
P55056
(P55056) Apolipoprotein C-IV precursor (Apo-CIV) (ApoC-IV)
14535.5
2
2
7
56%
0%
56%
more abundant in mild AD
gi|11386147
58094
0
0
0
1
2
1
4
0
4
−4
100%
gi|4505873
phospholipase D1,
124170
0
0
1
2
1
2
6
1
5
−4
67%
phophatidylcholine-specific
gi|13540475
serum amyloid A2
13491
0
0
1
1
1
2
5
1
4
−3
60%
gi|4504351
delta globin
16037
22
9
12
50
14
13
120
43
77
−34
28%
gi|4759050
ribosomal protein S6 kinase, 90 kDa,
83721
4
4
3
6
5
4
26
11
15
−4
15%
polypeptide 3
gi|4504489
histidine-rich glycoprotein
59559
5
10
13
8
11
19
66
28
38
−10
15%
precursor
gi|11761629
fibrinogen, alpha polypeptide
69739
25
44
18
46
54
19
206
87
119
−32
16%
isoform alpha preproprotein
gi|4557871
transferrin
77032
61
78
81
68
104
101
493
220
273
−53
11%
gi|10835095
serum amyloid A4, constitutive
14789
60
134
120
70
139
128
651
314
337
−23
4%
gi|49574514
matrix Gla protein
12336
0
0
0
3
0
1
4
0
4
−4
100%
gi|4506769
S100 calcium-binding protein A7
11440
0
1
0
1
1
4
7
1
6
−5
71%
gi|4502419
biliverdin reductase B (flavin
22101
0
0
0
1
0
3
4
0
4
−4
100%
reductase (NADPH))
gi|30794266
triggering receptor expressed on
32661
1
0
0
1
1
1
4
1
3
−2
50%
myeloid cells-like 1
gi|32698688
citron
231418
0
0
0
2
2
0
4
0
4
−4
100%
gi|28076869
serine (or cysteine) proteinase
44837
0
0
0
1
0
3
4
0
4
−4
100%
inhibitor, clade B (ovalbumin),
member 4
gi|40806175
diacylglycerol kinase, theta
101135
0
0
0
2
0
1
3
0
3
−3
100%
gi|38455402
lipocalin 2 (oncogene 24p3)
22571
0
0
0
0
1
1
2
0
2
−2
100%
gi|113417691
PREDICTED: hypothetical protein
24550
0
0
0
0
1
1
2
0
2
−2
100%
gi|21361470
chromosome 1 open reading frame
32112
0
0
0
1
1
0
2
0
2
−2
100%
48
gi|33286418
pyruvate kinase 3 isoform 1
57920
0
0
0
1
1
0
2
0
2
−2
100%
gi|66346708
membrane associated guanylate
136902
0
0
0
0
1
1
2
0
2
−2
100%
kinase, WW and PDZ domain
containing 1 isoform b
gi|24586657
myosin IIIA
186070
0
0
0
0
1
1
2
0
2
−2
100%
gi|11641247
chromosome 9 open reading frame
17200
0
0
0
1
1
0
2
0
2
−2
100%
19
gi|4507267
stanniocalcin 2 precursor
33230
0
0
0
1
0
1
2
0
2
−2
100%
gi|113426784
PREDICTED: similar to ribosomal
27009
0
0
0
0
1
1
2
0
2
−2
100%
protein S2
gi|19923424
myotubularin-related protein 9
63446
0
0
0
1
1
0
2
0
2
−2
100%
gi|4826663
core-binding factor, runt domain,
67115
0
0
0
1
0
1
2
0
2
−2
100%
alpha subunit 2; translocated to, 2
isoform MTGR1b
gi|21264361
mannan-binding lectin serine
75685
0
0
1
1
0
2
4
1
3
−2
50%
protease 2 isoform 1 precursor
gi|113419903
PREDICTED: similar to Neutrophil
10183
0
2
1
0
4
18
25
3
22
−19
76%
defensin 1 precursor (HNP-1) (HP-1)
(HP1) (Defensin, alpha 1)
gi|4557894
lysozyme precursor
16519
1
0
3
1
2
5
12
4
8
−4
33%
gi|38016947
complement component 5
188291
4
3
2
4
5
4
22
9
13
−4
18%
gi|38044288
gelsolin isoform a precursor
85680
2
5
2
5
7
2
23
9
14
−5
22%
gi|50363217
serine (or cysteine) proteinase
46720
21
25
17
30
25
31
149
63
86
−23
15%
inhibitor, clade A (alpha-1
antiproteinase, antitrypsin), member
1
gi|51339291
sterile alpha motif domain
184523
3
2
4
4
3
4
20
9
11
−2
10%
containing 9-like
gi|88853069
vitronectin precursor
54288
7
5
12
9
7
12
52
24
28
−4
8%
gi|50345296
complement component 4B
192735
12
12
10
13
16
10
73
34
39
−5
7%
preproprotein
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
sapiens]
more abundant in mild AD
P35542
SAA4_HUMAN
9.09E−04
14797.3
0.00
0.25
0.14
0.00
0.58
100%
61%
100%
0%
100%
100%
40%
100%
(P35542)
Serum
amyloid A-4
protein
precursor
(Constitutively
expres
P02766
TTHY_HUMAN
2.78E−13
15877.1
1.00
0.50
0.71
0.00
0.50
100%
18%
33%
100%
100%
100%
0%
33%
(P02766)
Transthyretin
precursor
(Prealbumin)
(TBPA) (TTR)
(ATTR)
P09874
PARP1_HUMAN
4.55E−04
112881.4
0.00
0.50
0.29
0.00
0.42
100%
19%
100%
0%
100%
100%
9%
100%
(P09874)
Poly [ADP-
ribose]
polymerase
1 (EC
2.4.2.30)
(PARP-1)
(AD
P01764
HV3C_HUMAN
4.40E−05
12574.2
0.00
0.75
0.43
0.00
0.42
100%
1%
100%
0%
100%
100%
29%
100%
(P01764) Ig
heavy chain
V-III region
VH26
precursor
Q9NPP6
Q9NPP6
7.65E−04
44758.1
0.33
0.50
0.43
0.00
0.33
100%
13%
20%
100%
100%
100%
20%
0%
(Q9NPP6)
Immunoglobulin
heavy
chain variant
(Fragment)
Q96MA6
Q96MA6
5.15E−04
54890.7
0.33
0.25
0.29
0.00
0.33
100%
8%
14%
100%
100%
100%
14%
0%
(Q96MA6)
Hypothetical
protein
FLJ32704
(Chromosome
9 open
reading
frame
Q9NYQ6
CELR1_HUMAN
1.30E−04
329276.7
0.33
0.50
0.43
0.00
0.33
100%
13%
20%
100%
100%
100%
20%
0%
(Q9NYQ6)
Cadherin
EGF LAG
seven-pass
G-type
receptor 1
precursor (
O60602
TLR5_HUMAN
2.76E−04
97663.4
0.00
0.00
0.00
0.00
0.33
100%
0%
0%
0%
100%
100%
100%
(O60602)
Toll-like
receptor 5
precursor
(Toll/interleukin-
1 recepto
Q15166
PON3_HUMAN
1.52E−05
39582.4
0.33
0.00
0.14
0.00
0.33
100%
40%
100%
100%
0%
100%
100%
0%
(Q15166)
Serum
paraoxonase/
lactonase 3
(EC 3.1.1.—)
more abundant in mild AD
This application claims priority to U.S. Provisional Application No. 60/855,749, filed Nov. 1, 2006, which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/023026 | 11/1/2007 | WO | 00 | 2/17/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60855749 | Nov 2006 | US |
