Detection of biomarkers with a multiplex quantitative ...

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Journal of Alzheimer’s Disease 9 (2006) 293–348 IOS Press

Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal fluid of patients with neurodegenerative disorders Fadi Abdia,∗∗ , Joseph F. Quinn b,∗∗ , Joseph Jankovic c, Martin McIntoshd , James B. Leverenz e, Elaine Peskinde, Randy Nixonb , John Nuttb , Katherine Chungb , Cyrus Zabetiane, Ali Samiie , Melanie Lina , Stephen Hattana , Catherine Pane , Yan Wange, Jinghua Jine , David Zhue , G. Jane Lie , Yan Liud , Dana Waichunas b, Thomas J. Montine e and Jing Zhang e,∗ a

Applied Biosystems, Framingham, MA, USA Oregon Health and Science University, Portland, OR, USA c Baylor College of Medicine, Houston, TX, USA d Fred Hutchison Cancer Research Center, Seattle, WA, USA e University of Washington School of Medicine, Seattle, WA, USA b

Abstract. Biomarkers are needed to assist in the diagnosis and medical management of various neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and dementia with Lewy body (DLB). We have employed a multiplex quantitative proteomics method, iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), in conjunction with multidimensional chromatography, followed by tandem mass spectrometry (MS/MS), to simultaneously measure relative changes in the proteome of cerebrospinal fluid (CSF) obtained from patients with AD, PD, and DLB compared to healthy controls. The diagnosis of AD and DLB was confirmed by autopsy, whereas the diagnosis of PD was based on clinical criteria. The proteomic findings showed quantitative changes in AD, PD, and DLB as compared to controls; among more than 1,500 identified CSF proteins, 136, 72, and 101 of the proteins displayed quantitative changes unique to AD, PD, and DLB, respectively. Eight unique proteins were confirmed by Western blot analysis, and the sensitivity at 95% specificity was calculated for each marker alone and in combination. Several panels of unique makers were capable of distinguishing AD, PD and DLB patients from each other as well as from controls with high sensitivity at 95% specificity. Although these preliminary findings must be validated in a larger and different population of patients, they suggest that a roster of proteins may be generated and developed into specific biomarkers that could eventually assist in clinical diagnosis and monitoring disease progression of AD, PD and DLB. Keywords: Alzheimer disease, biomarkers, Parkinson disease, proteomics

1. Introduction Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and dementia ∗ Corresponding author: Jing Zhang, MD, PhD, Division of Neuropathology, University of Washington School of Medicine, Box 359635 Harborview Medical Center, Seattle, WA 98104, USA. Tel.: +1 206 341 5245; Fax: +1 206 341 5249; E-mail: zhangj@ u.washington.edu. ∗∗ Authors who have contributed equally.

with Lewy body (DLB), are diagnosed primarily by clinical criteria, supported by laboratory investigations and functional neuroimaging analysis [3,25,46]. Although the diagnostic accuracy, verified by pathological examination, may reach 85–90% depending on the neurodegenerative disease, in many cases the diagnosis remains uncertain until death [20–22,28,35,41]. It is also noteworthy that it is common for patients with various neurodegenerative diseases to go undetected using current approaches [30]. Therefore, development

ISSN 1387-2877/06/$17.00 © 2006 – IOS Press and the authors. All rights reserved

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F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform

of biomarkers that may measure disease risk, presence, and progression is one of the main goals and challenges in research in neurodegenerative diseases. Ideally, an optimal biomarker should be precise, reliable, predictive, inexpensive, and should reflect the pathogenic mechanisms of neurodegenerative diseases. Presently, there are no established biomarkers that can be used to reliably confirm AD, PD or DLB or monitor their progression with high sensitivity and specificity. The usefulness of biomarkers would be markedly enhanced if they can detect disease at an early stage or even during the preclinical phase of the disease. Only limited success has been achieved in search for biochemical markers in body fluids, including plasma, urine, and cerebrospinal fluid (CSF), despite decades of research. This is clearly in part related to the heterogeneity of neurodegenerative diseases. Therefore, several biomarkers may be needed to detect subpopulations of patients [38]. The development of genomics, proteomics, and metabolomics has greatly enhanced our ability to discover additional markers for diagnosis of AD, PD and DLB and that may also shed light on disease pathogenesis. In fact, we, among others [6,9,10,19,40], have already started to use robust quantitative proteomics to identify unique markers related to aging [54] and AD [55] in CSF, an ideal source for discovering these markers because of its proximity to the brain. However, all of these proteomic studies, including our own, are limited, because none has taken other related and potentially confounding neurodegenerative diseases into consideration; in addition, very few studies have been performed using cases with pathological verification. This issue is particularly important in DLB, where despite continued refinements, the current clinical diagnostic criteria still have a relatively low sensitivity and specificity [35]. In this study, we employed an unbiased quantitative proteomic approach called iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification) [43] to label pre-fractionated human CSF followed by MudPIT (Multidimensional Protein Identification Technology) [27] prior to identification and quantification of CSF proteins with tandem mass spectrometry (MS/MS). This multiplex format allowed us to compare simultaneously the proteome of CSF in AD, PD, DLB patients and healthy controls. This analysis not only identified more than 1,500 CSF proteins, thereby greatly expanding our current knowledge about the human CSF proteome, but also detected 136, 72, and 101 proteins that displayed quantitative changes unique to

AD, PD, and DLB, respectively. Finally, the sensitivity at 95% specificity of each of eight confirmed markers or composite markers was calculated, demonstrating that the combination of several markers could distinguish among AD, PD and DLB patients not only from controls, but also from each other with high sensitivity at 95% specificity.

2. Materials and methods 2.1. Chemicals and antibodies All reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Antibody list: Apolipoprotein C1 (ApoC1; goat anti-human, Biodesign International, Kennebunkport, ME); ApoD (mouse monoclonal; Vision Biosystems, Norwell, MA); ApoH (rabbit polyclonal, Accurate Chemical & Scientific Corporation, Westbury, NY); calcium/calmodulindependent protein kinase IIB isoform 8 (Ca/CaMKIIB; rabbit polyclonal, Stratagen, Cedar Creek, TX); ceruloplasmin (sheep polyclonal, Abcam, Cambridge, MA), chromogranin B (rabbit polyclonal, Abcam); Cu/Zn superoxide dismutase (Cu/Zn SOD; mouse anti-human, Calbiochem, La Jolla, CA); β-fibrinogen (goat polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA); furin convertase (MON-148; mouse monoclonal, Alexis Biochemicals, San Diego, CA); α-1B-glycoprotein (A1BG; rabbit polyclonal, Aviva Systems Biology, San Diego, CA); haptoglobin (chicken polyclonal, Abcam), osteonectin (SPARC; mouse anti-human, Haematologic Technologies, Essex Junction, VT); semaphorin 7A (CDW108; mouse monoclonal, Chemicon International, Temecula, CA); T-cadherin (H-126; rabbit polyclonal, Santa Cruz Biotechnology); and vitamin D binding protein (VitD BP) or Gc-globulin (chicken polyclonal; GenWay Biotech, San Diego, CA). Secondary antibodies included rabbit anti-chicken IgG-HRP, rabbit antisheep IgG-HRP, rabbit anti-goat IgG-HRP, and goat anti-rabbit IgG-HRP (Sigma-Aldrich). Rabbit antimouse IgG-HRP was purchased from Abcam. 2.2. Patients Human Subject Institutional Review Boards of Baylor College of Medicine, Oregon Health and Science University (OHSU), and the University of Washington (UW) approved this study. All individuals underwent evaluation that consisted of medical history, physical and neurologic examinations, laboratory tests, and


neuropsychological assessment. Laboratory evaluation included complete blood count: serum electrolytes, blood urea nitrogen, creatinine, glucose, vitamin B12, and thyroid stimulating hormone; all results were within normal limits. A brief summary on inclusion and exclusion criteria is provided below for normal controls as well as patients with AD, PD or DLB. Demographic information is listed in Table 1 for all subjects/patients. Normal aged controls: The control subjects were compensated community volunteers in good health. Neuropsychological evaluation included: the MiniMental State Exam (MMSE) [13], Trail-Making Tests A and B [42], Clinical Dementia Rating Scale (CDR [37]), the Mattis and Coblentz Dementia Rating Scale score (DRS [32]) and the New York University (NYU) version of the Logical Memory II subscale (Immediate and Delayed Paragraph Recall) from the Wechsler Memory Scale – Revised [12]. Control subjects had no signs or symptoms suggesting cognitive decline or neurologic disease; all subjects had a MMSE score between 28 and 30; a CDR score of 0; and NYU paragraph recall scores (immediate and delayed) > 6. Exclusion criteria also included heavy cigarette smoking (more than 10 packs/year), alcohol use other than socially, and any psychotherapeutic use. Finally, it should be emphasized that although no pathological confirmation had been obtained in any of these subjects, all of them had been followed for approximately three years without demonstrating any symptoms or signs of neurological disorders, including mild cognitive impairment (MCI). AD: Patients were diagnosed with probable AD according to NINDS-ADRDA criteria confirmed by a clinical team consensus conference at the Oregon Aging and Alzheimer’s Disease Research Center and concurred by investigators at the UW Alzheimer’s Disease Research Center. Only patients with pathological confirmation of AD according to NIA-Reagan criteria (high), to the exclusion of Lewy body disease or vascular disease, were included in this study. CSF was collected during life and maintained at −70 ◦ C until analysis. The average time from CSF collected to autopsy was 2.4 years (also see Table 1). PD: Only clinically probable PD patients defined with NINDS criteria [15] were included. Patients were required to have three Group A signs,i.e. resting tremor, bradykinesia, rigidity and asymmetric onset, and have sustained response to levodopa or a dopamine (DA) agonist. Patients with the following features (Group B signs) were excluded: 1) prominent postural instability in the first three years after symptom onset, 2) freez-

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ing phenomenon in the first three years, 3) hallucinations unrelated to medications in the first three years, 4) dementia preceding motor symptoms or in the first year, 5) supranuclear gaze palsy (other than restriction of upward gaze) or slowing of vertical saccades, 6) severe, symptomatic dysautonomia unrelated to medications, and 7) documentation of a condition known to produce parkinsonism and plausibly connected to the patient’s symptoms (such as suitably located focal brain lesions or neuroleptic use within the past six months). Please note, like our control patients, all of these patients were still alive at the time of proteomic analysis; no pathological confirmation of PD had been obtained yet. Nonetheless, all patients included in this study had sustained response to DA drugs and there was no need to revise clinical diagnosis on any of these patients after follow-up evaluation when this manuscript was written. DLB: These patients were initially diagnosed with probable AD according to NINDS-ADRDA criteria, but each developed parkinsonism, fluctuating cognition, and visual hallucinations, characteristic of DLB, shortly after CSF was obtained, yielding a revised clinical diagnosis. Only patients with post-mortem confirmation of DLB were included in this study. All patients with DLB had co-existing changes of AD, a condition sometimes referred to as the Lewy Body Variant of AD [14]. CSF from this group of patients was the hardest to obtain, and thus this study was limited to include only five DLB cases. 2.3. Collection of CSF and quality control Following written informed consent, individuals were placed in the lateral decubitus position and the L45 interspace was infiltrated with 1% lidocaine. Lumbar puncture (LP) was performed with a 24G spinal needle. Individuals remained at bed rest for one hour following LP. All CSF for proteomic analysis was taken from the 15th to 25th ml collected to limit variations arising from rostral-caudal gradient. In addition, all LP was performed in the morning to limit potential circadian fluctuation of CSF proteins and metabolites. The protein concentration in CSF is relatively low compared to plasma (CSF:plasma =1/20–100), and in addition, the protein profiles in CSF are similar to those in plasma [5], suggesting that even a minor contamination of CSF with blood could significantly confound the interpretation of quantitative proteomic analysis of CSF. To minimize blood contamination in our CSF samples, only CSF samples with < 10 RBCs/ml and a serum:CSF ApoB (a protein not generated in CNS)

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F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 1 Characteristics of patients and age-matched controls

Control AD PD DLB

N

M:F

10 10 10 5

7:3 6:4 7:3 5:0

Age (Mean ± SD) 67 ± 6 72 ± 9 63 ± 7 69 ± 11

ratio > 6000 were included in this study. This approach has been utilized successfully in our previous CSF proteomics studies [54,55]. 2.4. Sample preparation before proteomic analysis Our previous experience has indicated that extensive analysis of well-characterized pooled samples is more productive than analyzing individual samples. This is largely due to the limitation of current MS technology, i.e. a low reproducibility when an identical sample is analyzed multiple times [54,57]. For example, if profiling is done with an individual sample, when a marker is identified in one individual (e.g., an AD patient) but not another, there is no way of telling whether it is due to the nature of the subject/patient or variation in ionization of MS unless an independent validation process is performed, which is not currently available in a high throughput manner. To circumvent this difficulty, in the last few years we have adopted the following strategy: discovering potential biomarkers with pooled samples (diseased vs. controls) with extensive chromatographic separation of peptides and multiple injections to reach the “bottom of the iceberg”. After potential biomarkers are identified, we confirm and/or validate them in individual samples to achieve information related to the sensitivity and specificity of each marker [54,55]. Hence, in the current study, in discovery phase we pooled CSF samples from 10 AD, 10 PD, 5 DLB, and 10 controls before proteomic analysis. The other issue related to CSF proteomics has to do with its unique profiles, i.e. overtly enriched in albumin and immunoglobulins (IgGs) [5] with a dynamic range of protein concentrations ∼10 9 as opposed to a dynamic range of ∼10 8 for typical cell lysates [8]. Because all current proteomic techniques are inheritably biased toward abundant proteins [53], fractionation of CSF is required before detailed proteomic analysis of CSF can be achieved. Thus, we followed a graduated organic fractionation approach that we recently developed to process CSF before standard MudPIT analysis of CSF proteins [54]. Briefly, pooled CSF was mixed with 1.5 volume of acetonitrile (ACN) first to generate the first

MMSE (Mean ± SD) 29 ± 1 13 ± 7 29 ± 1 20 ± 5

Time of CSF Tap to Autopsy (years) NA 2.4 ± 1.5 NA 2.5 ± 0.7

pellet (P1), and then the supernatant was further mixed with final 3.0 volume of ACN to generate the second pellet (P2) and a supernatant (S2), which was dialyzed with a porous (500 D) membrane to desalt. With this approach, more than 90% of albumin and IgGs are found in the first pellet [54]. It should be noted that we have examiend very carefully as to whether precipitation may variably affect the amount of proteins in each sample, demonstrating that significant variation in the amount of proteins due to precipitation is unlikely [54]. 2.5. iTRAQ labeling and two dimensional liquid chromatography Three fractions from each pooled CSF sample, i.e. P1, P2, and S2, were matched across all four groups of patients/subjects, forming three iTRAQ experiments. Briefly, 100 μg protein from each corresponding fraction (e.g., P1 fraction from AD, PD, DLB and controls) was digested in parallel with trypsin and then labeled with one of the four-iTRAQ TM reagents following the manufacturer’s instructions (Applied Biosystems or AB, Foster City, CA). Next, four samples labeled with iTRAQ reagents were combined (a total of 400 μg proteins), and loaded onto a strong cation exchange (SCX) PolySulfoethyl A TM column (2.1 × 200 mm, 5 micron, 300Å, Poly LC, Columbia, MD) that had been equilibrated in 5 mM KH 2 PO4 /25% ACN, pH 3.0 (buffer A) at a flow rate of 200 μl/min. Peptides were eluted by applying a linear gradient from 0 to 100% buffer B (5 mM KH 2 PO4 /600 mM KCl /25%ACN, pH 3.0). 11 fractions and 1 flow-through were collected from each sample and dried down in a SpeedVac (Thermo Savant, Holbrook, NY). SCX fractionated peptides from each sample were then dissolved in 0.5% trifluoroacetic acid (TFA) and separated using reverse phase (RP) chromatography. Nano-capillary liquid chromatography (LC) was performed using the LC Packings UltiMate TM with FamosTM autosampler and Switchos TM automated switching valve (LC Packings, Sunnyvale, CA). Samples were loaded onto a capillary precolumn cartridge (Dionex, Sunnyvale, CA). The trap column was


washed with mobile phase A containing 2% ACN and 0.1% TFA in HPLC water. The flow rate was set at 0.4 μl/min. The sample was then loaded onto a 15 cm X 100 μm ID Magic C18 3 μm, 100-angstrom packing capillary LC column (Michrome BioResources Inc., Auburn, CA). The gradient run was from 5% mobile phase B (80% ACN, 0.08% TFA in HPLC water) to 90% mobile phase B for 85 minutes. The eluted gradient was mixed with 7 mg/ml re-crystallized α-cyano4-hydroxycinnamic acid (Sigma) in 60% ACN, 2.6% (5 mg/ml) ammonium citrate with internal standard (4700 Mass Standard Kit, Applied Biosystems) and spotted onto a stainless steel MALDI plate with the ProbotTM (LC Packings). Samples were spotted at 5seconds intervals using a 24 × 24 array pattern for a total of 576 spots per plate. In total, 36 MALDI plates were spotted and analyzed by a 4700 Proteomic System (see below).

2.6. Tandem MS analysis and protein identification Quantitative MS/MS analysis was carried out using the 4700 Proteomics Analyzer with TOF/TOF OpticsTM (Applied Biosystems). MS reflector positive ion mode with automated acquisition of 800–4000 m/z range was used with 1000 shots per spectrum. A maximum of 15 peaks were selected per spot, with a minimum signal-noise (S/N) ratio of 50 and cluster area of 500. More than 36000 precursors were selected and submitted for MS/MS, where a positive ion mode with collision induced-dissociation (CID) cell and 1kV collision energy were used, and 3000 shots were accumulated per spectrum. A total of 576 MS and more than 1200 MS/MS spectra were acquired for each spotted plate. Identification of proteins was achieved using Mascot (Matrix Science, Boston, MA) algorithm and searched against the International Protein Index (IPI; Version 3.01), a database also used in one of our recent studies of human CSF [52]. In addition, protein identification was determined with a newer version of the IPI database (3.10) as well as with the Celera Discovery SystemTM database (20050302) that is typically used by the 4700 Proteomic System. Protein quantification was achieved by averaging iTRAQ ratios of all peptides identified; normalization, using a Gaussian distribution with median of 1 when all peptides were considered between control and experimental groups, was performed after iTRAQ ratios were calculated.

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2.7. Western blot Western blot analysis was performed as described previously [2] with minor modifications. In brief, equal amounts of human CSF proteins (and equal volumes as well for pooled samples) were run on SDS/PAGE TrisHCl Criterion Gels (Bio-Rad Laboratories, Hercules, CA) under reducing conditions, transferred to PVDF membranes (Bio-Rad), blocked, and probed overnight at 4◦ C with primary antibodies of ApoC1 (1:2000), ApoD (1:10000), ApoH (1:1000), A1BG (1:10000), chromogranin B (1:10000), Ca/CaMKIIB (1:500), ceruloplasmin (1:2000), β-fibrinogen (1:2000), furin (1:5000), haptoglobin (1:2000), semaphorin 7A (1:500), SPARC (1:5000), Cu/Zn-SOD (1:10000), Tcadherin (1:250), or VitD BP (1:4000). The secondary antibodies were added, and detected by enhanced chemiluminescence or by ECL plus western blotting detection system (Amersham Biosciences, NJ). Relative levels of each protein were quantified by measuring optical densities (OD) of the corresponding bands compared to a pooled sample containing all cases. Protein concentration of the CSF was determined by the Bradford method with bovine serum albumin (Pierce, IL) as the standard. 2.8. Quantifying the diagnostic ability of candidate markers Quantifying the diagnostic ability of a single marker: The performance of each of the eight confirmed candidate markers was evaluated both graphically and statistically with receiver operating characteristic (ROC) curve methods. ROC curves associate the sensitivity of a diagnostic test to the entire range of the possible false positive rate (FPR). The FPR is equal to one minus the test specificity. The area under the ROC curve (AUC) indicates the average sensitivity of a marker over the entire ROC curve. The sensitivity of each marker was also computed from the ROC curve at 95% specificity. Establishing statistical significance of a single marker was performed by the Wilcoxon rank-sum test, which evaluates the significance of the entire ROC curve. To aid interpretation of our data when comparing markers, raw data was transformed with the natural log so its behavior among healthy subjects more accurately reflected a normal distribution with a mean of 0 and unit standard deviation [34]. Standardization of the markers, which leaves the ROC curves unchanged, also facilitates the comparison of two different markers (see below) because the units of measurement are now

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similar, i.e. the number of standard deviations above the average normal subject. Combining markers: After the eight candidate markers were ranked based on the sensitivity at 95% specificity, p-value from Wilcoxon rank-sum test, and the AUC value, the top five individual markers were chosen for calculation of a composite marker (CM), which summarizes all the markers together into a single diagnostic marker summary. This was accomplished by evaluating a linear combination, which can be easily interpreted, estimated by Logistic regression. Logistic regression has several theoretical properties that make it convenient for applied biomarker research [33]. For instance, the P values for each marker in the composite evaluate whether it significantly increases the “distance” between cases and controls. If the model is correctly specified, the sensitivity of the resulting CM is maximized at all specificities simultaneously, although the theoretically correct model cannot ever be known in practice. After establishing significance, we then examine resulting ROC curves to evaluate the quality of the composite marker.

3. Results 3.1. CSF proteome Using pooled, well characterized CSF samples and multidimensional peptide separation techniques, followed by 4700 TOF-TOF analysis, we were able to identify and quantify a total of 1,539 proteins (Appendix I and II). Of these, 793 were identified from the MS/MS spectrum of a single peptide (“single hit”) (Appendix II) and are therefore judged to be less reliably identified than those proteins identified with multiple peptide tandem mass spectra (Appendix I). All protein identification was based on meeting the criteria of having at least one peptide whose individual composite score was above the 95% confidence interval threshold (p < 0.05) and also identified as the top-ranked matching sequence for that spectrum. In addition, for proteins identified by a single peptide, additional criteria were applied, including: 1) the Mascot ion score had to be greater than 30 and 2) all peptides had to contain tryptic digestion end. These criteria were applied because it has been estimated by several groups of investigators that false-positive (FP) rate is typically less than 1% for protein identification when these criteria are used [24, 51].

When the list of current protein identification was compared to our previous analysis of human CSF, where close to 1,000 proteins were identified [52] using the same database, 449 of those proteins were identified again in our current study. To state it differently, 1,090 new proteins were identified in the current study, thereby increasing our total identified CSF proteins to 1,882. Of note was also the observation that 51 proteins identified previously by a single peptide were now identified by more than two peptides. Examples included testican-1 precursor, ApoM, neuroligin 2 precursor, xylosyltransferase I, and sortilin 1 preprotein. On the other hand, 90 proteins identified previously by more than two peptides were now identified by only a single peptide. These included cathepsin L precursor, collagen alpha 1(III) chain precursor, Mn-SOD, mitochondrial precursor, gelsolin precursor, and peroxiredoxin 2. Thus, the proteins identified by more than two peptides were 1,097 when all of our studies are combined. We are fully aware that guidelines of Human Proteome Organization (HUPO) suggest that more than two peptides are needed for more confident protein identification, and in fact, this is why we have kept single hits in a separate Appendix for future reference in current, as well as in all of our previous CSF studies [52,54,55]. Classification of the 1,539 proteins identified in this study, shown in Fig. 1, is based on a modified scheme that we have developed in our previous publications [54,55]; it includes neuronal activities/signal transduction, cell cycle/death, cell structure/motility/transport/traffic, metabolism, extracellular matrix/cell adhesion, immunity/defense, and unknown functions. However, as demonstrated in Table 2, the database used for protein identification can have a significant role in not only the number of proteins identified but also the classifications of proteins [52]. As a result, we used IPI 3.01 (a database used in our recent CSF study) rather than a more updated version IPI 3.10 in this study to make sure our data can be compared meaningfully. Still, to further illustrate the contribution of databases on the outcome of protein identification, the identical MS data generated by 10 plates of 1.5P fraction were searched against the updated IPI version (3.10) as well as the newest Celera Discovery System (CDS) database (20050302) provided by Applied Biosystems, the manufacturer of the 4700 proteomic station. The results for database comparison are listed in Table 2 with several major points noted below. First, the overlap was only 26.0% between IPI 3.01 and CDS database if common protein names were used, but improved to 86.8% when peptides were con-


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Table 2 Identification of proteins with the same MS data against different databases Proteins Identified IPI (3.01) IPI (3.10) Celera Common names Two peptides Single peptide Peptides regardless of protein name

567 250 317 1584

559 291 268 1759

782 569 213 1506

Overlap IPI Celera vs. 3.10 vs. 3.01 IPI 3.01 62.9% 26.0% 65.4% 30.5% 58.9% 23.2% 96.7% 86.8%

Neuronal Activities/Signal Transduction Cell Cycle/Death Cell Structure/Motility/Transport/Traffic 18%

21%

Metabolism Extracellular Matrix /Cell Adhesion Im munity/Defense Unknow n

6% 4%

8%

15%

28%

Fig. 1. Pie chart depicting the 1,539 proteins characterized by nano-LC-MALDI-TOF-TOF. Functional classification of a given protein was based on the one that is best known, although typically multiple functions may have been associated with that particular protein. Notably, a significant portion of the proteome is novel without known functions.

sidered regardless of protein names. Second, the overlap was much higher between two different versions of IPI database; and, consistent with our previous results, the overlap was higher for proteins identified by more than two peptides than those by single peptide. Finally, although the IPI database appears to be maturing, the difference in protein identification between 3.01 and an earlier version [52] vs. 3.01 and 3.10 was about the same, i.e. a change in the database when an identical MS data set was used resulted in about a 10% difference in protein identification. 3.2. Changes in CSF proteome associated with AD, PD, and DLB Individual quantification of the identified peptides were based on the individual ratios from signature ion peak areas of the iTRAQ reagent tags of the identified peptides from AD, PD and DLB samples compared

with the healthy individuals’ signature ion peak areas. The iTRAQ ratios of all the peptides for each protein were grouped and averaged together to give protein level modulations ratios for 1,520 identified proteins. Modulated proteins were found to be involved in several biological processes such as neural activities/signal transductions, cell cycle/death, cell structure/motility/transport/traffic, metabolism, extracellular matrix/adhesion, and immunity/defense (Table 3). Some of these modulations exceeded two or three folds up or down. As the first step towards selecting candidate proteins for further study, we chose to define the changes with more than 50% increase or decrease as significant. Changes that were less than < 20% and > 20% but less than 50% were defined as having unlikely and uncertain significance. With these criteria, AD, PD, and DLB patients had a total of 388, 282, and 380 proteins that displayed significant changes from controls. Next, we further narrowed our focus on the protein markers that were

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F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 3 Proteins with changes unique to AD, PD or DLB The assignment of function of each protein is putative, as most, if not all, proteins have multiple functions IPI #

iTRAQ Ratios PD DLB

Protein Name Proteins unique to AD and identified by 2 or more peptides Neuronal Activities/Signal Transduction Brain abundant, membrane attached signal protein 1 Brain-derived neurotrophic factor BDNF1 Cell growth regulator with EF hand domain 1 Chromogranin A Chromogranin B Insulin-like growth factor binding protein 5 precursor Neurexin 1-alpha precursor Neuronal pentraxin I precursor Neuronal pentraxin receptor PLXDC2 protein PREDICTED: lunatic fringe homolog Prostatic binding protein Protein KIAA0494 Reticulocalbin 2 precursor Secretogranin I precursor Secretogranin III precursor Splice isoform 2 of insulin-like growth factor II precursor Splice isoform 3 of calcium/calmodulin-dependent protein kinase type II beta chain TBC1 domain family member 10

AD

IPI00299024 IPI00336003 IPI00337548 IPI00419463 IPI00006601 IPI00029236 IPI00442299 IPI00220562 IPI00031289 IPI00073777 IPI00455739 IPI00219446 IPI00006130 IPI00029628 IPI00006601 IPI00292071 IPI00215977 IPI00219165 IPI00011167

0.39 0.60 0.77 0.58 0.49 1.51 0.47 0.46 0.64 0.67 0.47 0.46 0.66 1.25 0.65 0.65 1.57 0.64 1.25

↓↓ ↓↓ ↓ ↓↓ ↓↓ ↑↑ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↑ ↓↓ ↓↓ ↑↑ ↓↓ ↑

0.92 0.84 1.67 0.91 1.70 0.79 2.14 0.99 1.13 0.83 1.03 0.91 0.95 0.99 1.06 1.03 1.07 0.85 1.03

NC NC ↑↑ NC ↑↑ ↓ ↑↑ NC NC NC NC NC NC NC NC NC NC NC NC

1.34 0.91 1.12 0.83 1.09 0.85 1.17 1.43 1.11 1.26 1.79 0.84 0.96 0.87 0.84 0.98 1.11 1.05 1.04

Cell Cycle/Death Cadherin-13 precursor Glucosidase II beta subunit precursor Golgi autoantigen, golgin subfamily B member 1 Latent transforming growth factor beta binding protein 2 Neuroblastoma suppressor of tumorigenicity 1 precursor Splice isoform 1 of SWI/SNF-related, matrix associated, actin-dependent regulator

IPI00024046 IPI00026154 IPI00004671 IPI00465145 IPI00013299 IPI00220119

0.55 0.64 0.47 1.71 1.55 1.61

↓↓ ↓↓ ↓↓ ↑↑ ↑↑ ↑↑

1.11 1.11 2.14 1.08 1.13 0.96

NC NC ↑↑ NC NC NC

0.84 NC 1.07 NC 1.17 NC 0.82 ↓ 1.09 NC 0.71 ↓

Cell Structure/Motility/Transport/Traffic 107 kDa protein Alpha-1-acid glycoprotein 1 precursor Apolipoprotein A-II precursor Apolipoprotein C-1 precursor Apolipoprotein D precursor Apolipoprotein E precursor Apolipoprotein H Collagen alpha 2(I) chain precursor Divalent cation tolerant protein CUTA Golgi phosphoprotein 2 Hypothetical protein FLJ25530 Hypothetical protein MOT8 KIAA1291 protein Ly-6/neurotoxin-like protein 1 precursor Neurofascin isoform 2 Receptor-type tyrosine-protein phosphatase-like N precursor Sortilin 1, preproprotein Tetranectin precursor

IPI00476999 IPI00022429 IPI00021854 IPI00021855 IPI00006662 IPI00021842 IPI00298828 IPI00304962 IPI00034319 IPI00171411 IPI00167215 IPI00001399 IPI00413206 IPI00289058 IPI00477942 IPI00004440 IPI00383591 IPI00009028

0.28 2.49 1.99 1.18 1.63 0.77 1.22 1.53 0.82 2.08 0.59 2.18 1.76 0.48 0.59 0.33 1.54 0.63

↓↓ ↑↑ ↑↑ NC ↑↑ ↓ ↑ ↑↑ ↓ ↑↑ ↓↓ ↑↑ ↑↑ ↓↓ ↓↓ ↓↓ ↑↑ ↓↓

0.85 0.71 0.66 0.99 0.96 1.14 0.66 1.02 0.96 0.89 0.99 0.65 1.14 1.03 0.87 0.89 1.06 0.98

NC ↓ ↓↓ NC NC NC ↓↓ NC NC NC NC ↓↓ NC NC NC NC NC NC

0.99 1.02 1.06 0.48 1.16 0.93 0.84 1.18 2.07 0.77 0.87 1.08 0.70 0.94 0.91 0.85 0.65 1.35

NC NC NC ↓↓ NC NC NC NC ↑↑ ↓ NC NC ↓ NC NC NC ↓↓ ↑

Metabolism Angiotensinogen precursor Enolase 2 Hect domain and RLD 4 Hypothetical protein DKFZp686B0286 Kallikrein 6 precursor Phosphatidylcholine-sterol acyltransferase precursor ProSAAS precursor Superoxide dismutase 1, soluble Transcription elongation regulator 1

IPI00032220 IPI00216171 IPI00333067 IPI00465248 IPI00023845 IPI00022331 IPI00002280 IPI00218733 IPI00247871

0.61 0.57 1.62 0.57 1.90 0.60 0.65 0.58 2.33

↓↓ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↓↓ ↓↓ ↑↑

1.02 1.00 0.67 1.00 0.80 1.08 1.07 1.06 0.94

NC NC ↓↓ NC ↓ NC NC NC NC

1.70 1.32 0.90 1.32 0.86 0.91 1.11 1.68 0.71

↑↑ ↑ NC ↑ NC NC NC ↑↑ ↓

↑ NC NC NC NC NC NC ↑ NC ↑ ↑↑ NC NC NC NC NC NC NC NC


301

Table 3, continued Protein Name Vacuolar ATP synthase subunit S1 precursor

iTRAQ Ratios IPI # AD PD DLB IPI00020430 0.50 ↓↓ 0.96 NC 0.84 NC

Extracellular Matrix/Cell Adhesion Cochlin precursor Matrix Gla-protein precursor Spondin 1 precursor

IPI00012386 1.22 ↑ 0.62 ↓↓ 1.14 NC IPI00028714 1.51 ↑↑ 0.90 NC 0.97 NC IPI00171473 1.76 ↑↑ 0.90 NC 0.93 NC

Immunity/Defense 24 kDa protein AMBP protein precursor Complement C2 precursor Cytokine-like protein C17 precursor Fibrinogen beta chain precursor HLA class I histocompatibility antigen, B-27 alpha chain precursor HLA class I histocompatibility antigen, E alpha chain precursor Myosin-reactive immunoglobulin heavy chain variable region

IPI00479531 IPI00022426 IPI00303963 IPI00032876 IPI00298497 IPI00471986 IPI00010362 IPI00007893

3.02 2.09 0.59 1.65 1.55 1.52 1.28 1.53

↑↑ ↑↑ ↓↓ ↑↑ ↑↑ ↑↑ ↑ ↑↑

0.70 0.88 0.89 0.83 0.97 1.13 1.08 0.86

↓ NC NC ↓ NC NC NC NC

1.05 1.10 1.07 1.10 1.14 1.06 1.18 1.04

NC NC NC NC NC NC NC NC

Unknown 132 kDa protein Haptoglobin precursor Hypothetical protein Hypothetical protein DKFZp566O224 Hypothetical protein DKFZp761H2024 Hypothetical protein FLJ33620 Hypothetical protein FLJ33674 Hypothetical protein PSEC0072 Inter-alpha-trypsin inhibitor heavy chain H1 precursor KIAA1318 protein Uveal autoantigen VPS10 domain-containing receptor SORCS3 precursor

IPI00477893 IPI00478493 IPI00165652 IPI00383815 IPI00185662 IPI00216853 IPI00301019 IPI00168884 IPI00292530 IPI00002353 IPI00173359 IPI00010381

1.48 1.97 0.54 1.76 0.32 1.56 0.66 0.65 1.55 1.23 0.61 0.66

↑ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↓↓ ↑↑ ↑ ↓↓ ↓↓

0.49 1.23 0.90 0.76 0.93 1.15 1.18 1.04 0.98 0.90 1.00 1.08

↓↓ ↑ NC ↓ NC NC NC NC NC NC NC NC

1.01 1.14 0.94 1.16 1.19 1.06 1.17 0.87 0.98 1.17 1.10 1.05

NC NC NC NC NC NC NC NC NC NC NC NC

Proteins unique to AD and identified by single peptide Neuronal Activities/Signal Transduction Bone morphogenetic protein 15 precursor IPI00001485 G protein-coupled sphingolipid receptor IPI00015343 IL-17RC IPI00303074 Interleukin-1 receptor-associated kinase-like 2 IPI00304986 Metallothionein-III IPI00016666 Neural proliferation differentiation and control protein-1 precursor IPI00299699 Potassium voltage-gated channel subfamily KQT member 3 IPI00012857 Putative 4 repeat voltage-gated ion channel IPI00217996 Splice Isoform 1 of protachykinin 1 precursor IPI00023571 Splice isoform 2 of UDP-N-acetylglucosamine–peptide N-acetylglucosaminyltransferase 11 IPI00219856 Voltage-dependent calcium channel gamma-6 subunit IPI00011072

0.64 0.62 0.63 0.58 0.47 0.51 0.62 0.64 0.43 1.62 1.53

↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↑↑ ↑↑

1.06 0.86 0.96 1.00 0.95 1.01 0.96 1.26 1.01 0.87 0.94

NC NC NC NC NC NC NC ↑ NC NC NC

1.08 0.96 0.87 0.88 0.97 1.05 0.90 0.91 0.87 1.01 1.20

NC NC NC NC NC NC NC NC NC NC NC

Cell Cycle/Death AlphA 1 type XIII collagen isoform 3 Integral membrane protein 2B

IPI00375409 1.26 ↑ 0.63 ↓↓ 0.94 NC IPI00031821 1.80 ↑↑ 0.94 NC 1.08 NC

Cell Structure/Motility/Transport/Traffic Actin, aortic smooth muscle Cohesin subunit SA-1 Hepatocellular carcinoma associated protein TB6 SAA1 protein SAYY8238 Splice isoform 1 of hpaII tiny fragments locus 9c protein Splice isoform 2 of development and differentiation-enhancing factor 2 Splice isoform 2 of putative polypeptide N-acetylgalactosaminyltransferase-like protein TRIF-related adapter molecule

IPI00008603 IPI00025158 IPI00293898 IPI00452748 IPI00432771 IPI00337307 IPI00409613 IPI00456715 IPI00329281

Metabolism ADAM 10 precursor Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase Cytochrome P450 1A1

IPI00013897 1.54 ↑↑ 0.99 NC 0.91 NC IPI00000138 0.49 ↓↓ 1.02 NC 0.91 NC IPI00218839 0.55 ↓↓ 1.03 NC 0.88 NC

1.72 1.64 1.57 1.86 0.59 0.65 1.96 0.66 1.87

↑↑ ↑↑ ↑↑ ↑↑ ↓↓ ↓↓ ↑↑ ↓↓ ↑↑

1.05 0.91 1.01 0.78 0.86 0.95 0.93 1.07 0.94

NC NC NC ↓ NC NC NC NC NC

1.09 1.12 1.05 0.91 1.05 1.14 1.03 0.89 1.03

NC NC NC NC NC NC NC NC NC

302

F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 3, continued

Protein Name DNA-directed RNA polymerase I largest subunit Heat shock 10 kDa protein 1 (chaperonin 10) Mosaic serine protease PPIB protein Splice isoform 2 of insulin receptor precursor Transcriptional activator SRCAP Zinc finger protein 95 homolog ZNF627 protein

IPI # IPI00031960 IPI00220362 IPI00012505 IPI00419262 IPI00220325 IPI00009101 IPI00032316 IPI00029023

AD 0.59 1.71 1.84 0.55 0.59 0.59 2.80 3.04

iTRAQ Ratios PD DLB ↓↓ 0.89 NC 1.32 ↑↑ 1.19 NC 0.97 ↑↑ 1.12 NC 1.06 ↓↓ 1.03 NC 0.88 ↓↓ 0.87 NC 0.86 ↓↓ 0.94 NC 0.84 ↑↑ 1.17 NC 1.19 ↑↑ 0.00 ↓↓ 0.00

↑ NC NC NC NC NC NC ↓↓

Extracellular Matrix/Cell Adhesion Inhibin beta A chain precursor KIAA1730 protein PREDICTED: KIAA0527 protein PREDICTED: odz, odd Oz/ten-m homolog 3 Splice isoform 1 of ADAMTS-16 precursor

IPI00028670 IPI00155199 IPI00297224 IPI00398020 IPI00386697

0.66 1.63 0.66 1.91 0.64

↓↓ ↑↑ ↓↓ ↑↑ ↓↓

NC ↓↓ NC NC NC

Immunity/Defence Pregnancy-specific beta-1-glycoprotein 8 precursor

IPI00334256 2.79 ↑↑ 1.07 NC 0.88 NC

Unknown 141 kDa protein 15 kDa protein 25 kDa protein Antigen MLAA-20 C1orf40 protein DJ977L11.1 FLJ00199 protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein DKFZp434A2017 Hypothetical protein DKFZp666G229 Hypothetical protein DKFZp686A06175 Hypothetical protein DKFZp781A0122 Hypothetical protein PSEC0200 KARCA1 protein Kelch/ankyrin repeat containing cyclin A1 interacting protein Optic atrophy 1 isoform 4 PREDICTED: hypothetical protein XP 374046 PREDICTED: similar to melanoma antigen, family A, 10 Similar to expressed sequence AI593442

IPI00478948 IPI00413387 IPI00477989 IPI00447178 IPI00304374 IPI00478622 IPI00291731 IPI00032525 IPI00333324 IPI00470772 IPI00295380 IPI00470388 IPI00478616 IPI00470805 IPI00166392 IPI00168703 IPI00449308 IPI00107749 IPI00397059 IPI00455972 IPI00217781

1.64 1.51 2.38 1.56 0.63 1.55 0.83 1.66 1.57 0.67 1.67 0.60 1.58 0.66 1.54 5.81 5.65 2.20 0.66 0.64 0.45

↑↑ ↑↑ ↑↑ ↑↑ ↓↓ ↑↑ ↓ ↑↑ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↑↑ ↑↑ ↑↑ ↑↑ ↓↓ ↓↓ ↓↓

0.91 0.93 1.00 1.10 0.96 0.61 1.15 1.09 1.13 1.10 1.01 1.09 1.13 1.14 1.00 1.06 1.09 1.02 1.04 1.06 1.18

NC NC NC NC NC ↓↓ NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

1.12 1.10 1.13 1.14 0.87 0.94 1.15 1.09 1.01 1.00 1.08 1.12 0.85 1.06 1.11 0.57 0.76 1.05 0.84 1.08 0.86

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC ↓↓ ↓ NC NC NC NC

Proteins unique to PD and identified by 2 or more peptides Neuronal Activities/Signal Transduction Amyloid-like protein 1 precursor Cell growth regulator with EF hand domain 1 Chromogranin B C-type natriuretic peptide precursor DA141H5.1 Insulin-like growth factor binding protein 5 precursor Neurexin 1-alpha precursor Prion protein Protein tyrosine phosphatase, non-receptor type substrate 1 precursor Reticulon 4, isoform D Serine/threonine-protein kinase PLK2 Splice isoform 1 of basigin precursor Splice isoform 1 of lysosomal trafficking regulator Splice isoform 7 of amyloid beta A4 protein precursor

IPI00020012 IPI00337548 IPI00006601 IPI00012075 IPI00478414 IPI00029236 IPI00442299 IPI00382843 IPI00332887 IPI00335276 IPI00302787 IPI00218019 IPI00017094 IPI00219187

1.11 0.77 0.49 0.87 0.98 1.51 0.47 1.00 1.00 0.98 0.91 0.98 1.05 0.93

NC ↓ ↓↓ NC NC ↑↑ ↓↓ NC NC NC NC NC NC NC

0.66 1.67 1.70 1.54 1.31 0.79 2.14 1.57 1.52 0.50 0.44 1.21 0.36 1.57

↓↓ ↑↑ ↑↑ ↑↑ ↑ ↓ ↑↑ ↑↑ ↑↑ ↓↓ ↓↓ ↑ ↓↓ ↑↑

0.91 1.12 1.09 1.00 0.55 0.85 1.17 1.00 0.71 0.99 1.06 0.67 0.95 0.97

NC NC NC NC ↓↓ NC NC NC ↓ NC NC ↓↓ NC NC

Cell Cycle/Death Golgi autoantigen, golgin subfamily B member 1 Heparin-binding EGF-like growth factor precursor

IPI00004671 0.47 ↓↓ 2.14 ↑↑ 1.17 NC IPI00012948 0.84 NC 0.64 ↓↓ 0.84 NC

1.14 1.18 1.04 0.72 0.87

NC NC NC ↓ NC

1.06 0.66 0.98 0.99 0.93


303

Table 3, continued iTRAQ Ratios PD DLB

Protein Name Cell Structure/Motility/Transport/Traffic Alpha-1-acid glycoprotein 1 precursor Apolipoprotein A-II precursor Apolipoprotein C-I precursor Apolipoprotein C-III precursor Apolipoprotein H Apolipoprotein M Hook homolog 3 Hypothetical protein MOT8 KIAA1265 protein KRT8 protein Retinol binding protein 4, plasma Selenoprotein M precursor

IPI #

AD

IPI00022429 IPI00021854 IPI00021855 IPI00021857 IPI00298828 IPI00030739 IPI00031768 IPI00001399 IPI00008085 IPI00418411 IPI00479848 IPI00103471

2.49 1.99 1.59 0.89 1.22 1.11 0.89 2.18 1.08 1.15 1.02 1.06

↑↑ ↑↑ ↑↑ NC ↑ NC NC ↑↑ NC NC NC NC

0.71 0.66 0.78 0.57 0.66 0.60 0.63 0.65 0.55 0.64 0.63 0.66

↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓ ↓↓

1.02 1.06 1.05 1.28 0.84 0.91 1.01 1.08 1.66 0.85 1.08 0.93

NC NC NC ↑ NC NC NC NC ↑↑ NC NC NC

Metabolism Ceruloplasmin precursor Cystatin C precursor Hect domain and RLD 4 Kallikrein 6 precursor Prothrombin precursor Pyruvate kinase 3 isoform 2 Selenium binding protein 1 Vitamin D-binding protein precursor


0.93 0.88 1.62 1.90 1.08 1.11 1.16 1.06

NC NC ↑↑ ↑↑ NC NC NC NC

0.65 0.61 0.67 0.80 0.57 1.80 1.56 0.83

↓↓ ↓↓ ↓↓ ↓ ↓↓ ↑↑ ↑↑ ↓↓

0.84 0.89 0.90 0.86 0.87 1.13 1.19 1.01

NC NC NC NC NC NC NC NC

Extracellular Matrix/Cell Adhesion Cochlin precursor Extracellular matrix protein 1 Splice isoform 3 of integrin alpha-7 precursor

IPI00012386 1.22 ↑ 0.62 ↓↓ 0.96 NC IPI00006969 0.97 NC 0.53 ↓↓ 1.05 NC IPI00220749 0.99 NC 0.64 ↓↓ 0.98 NC

Immunity/Defense 21 kDa protein 24 kDa protein CD99L2 protein Polymeric-immunoglobulin receptor precursor

IPI00477336 IPI00479531 IPI00434755 IPI00004573

Unknown 132 kDa protein Hypothetical protein DKFZp566O224 PREDICTED: G2 protein

IPI00477893 1.48 ↑ 0.49 ↓↓ 1.01 NC IPI00383815 1.76 ↑↑ 0.76 ↓ 1.16 NC IPI00176482 0.92 NC 1.28 ↑ 0.58 ↓↓

Proteins unique to PD and identified by single peptide Neuronal Activities/Signal Transduction Activating receptor pilrbeta IPI00186781 PREDICTED: KIAA1337 protein IPI00002283 PREDICTED: similar to 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated IPI00376589 protein) Rho-GTPase activating protein 10 IPI00169307

1.19 3.02 1.11 1.12

NC ↑↑ NC NC

1.59 0.70 1.63 0.15

↑↑ ↓ ↑↑ ↓↓

1.17 NC 1.05 NC 0.42 ↓↓ 1.52 ↑↑

0.97 NC 0.72 ↓ 1.62 0.97 NC 0.64 ↓↓ 1.49 0.84 NC 1.53 ↑↑ 0.62

↑↑ ↑ ↓↓

1.00 NC 5.99 ↑↑ 0.48

↓↓

Cell Cycle/Death Alpha 1 type XIII collagen isoform 3

IPI00375409 1.26

Cell Structure/Motility/Transport/Traffic ATP-binding cassette, sub-family A, member 1 Hypothetical protein DKFZp434P097 Hypothetical protein FLJ32842 Putative 4 repeat voltage-gated ion channel Ribonuclease 4 precursor Ribosomal protein L3-like SAA1 protein Splice isoform 1 of transcription factor E2-alpha


1.05 0.89 0.97 0.91 1.08 0.89 0.91 1.74

NC NC NC NC NC NC NC ↑↑

Metabolism Metabotropic glutamate receptor 3 precursor

IPI00478165 0.86 NC 0.51 ↓↓ 2.43

↑↑

Extracellular matrix/Cell Adhesion Laminin gamma-1 chain precursor

IPI00298281 0.93 NC 1.37

1.10 1.10 0.84 0.64 0.88 1.10 1.86 0.84

↑ NC NC NC ↓↓ NC NC ↑↑ NC

0.63 ↓↓ 0.94 NC 1.57 0.55 0.46 1.26 1.68 0.55 0.78 0.72

↑↑ ↓↓ ↓↓ ↑ ↑↑ ↓↓ ↓ ↓

↑

0.60

↓↓

304

F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 3, continued iTRAQ Ratios AD PD DLB 1.14 NC 0.57 ↓↓ 1.55 ↑↑ 1.91 ↑↑ 0.72 ↓ 0.99 NC 1.18 NC 0.64 ↓↓ 1.04 NC

Protein Name Mammalian ependymin related protein 1 PREDICTED: odz, odd Oz/ten-m homolog 3 Splice Isoform 2 of integrin alpha-7 precursor

IPI # IPI00259102 IPI00398020 IPI00220748

Immunity/Defense Ig kappa chain V-I region HK102 precursor

IPI00478600 1.10 NC 1.53 ↑↑ 0.87 NC

Unknown 97 kDa protein DJ977L11.1 HRPE773 Hypothetical protein Hypothetical protein FLJ16127 Hypothetical protein FLJ46550 OTTHUMP00000021593

IPI00472544 IPI00478622 IPI00060800 IPI00470620 IPI00442326 IPI00443682 IPI00374531

0.00 1.55 0.99 1.08 1.13 0.84 0.00

↓↓ ↑↑ NC NC NC NC ↓↓

2.75 0.61 0.44 0.55 0.63 1.69 2.75

↑↑ ↓↓ ↓↓ ↓↓ ↓↓ ↑↑ ↑↑

0.00 0.94 1.78 1.12 0.93 0.91 0.00

↓↓ NC ↑↑ NC NC NC ↓↓

Proteins unique to DLB and identified by 2 or more peptides Neuronal Activities/Signal Transduction 110 kDa protein Brain abundant, membrane attached signal protein 1 Cocaine- and amphetamine-regulated transcript protein precursor DA141H5.1 Latent transforming growth factor-beta binding protein 4 Neurexophilin 4 Neuronal pentraxin I precursor Parvalbumin PLXDC2 protein PREDICTED: lunatic fringe homolog Proenkephalin A precursor Protein tyrosine phosphatase, non-receptor type substrate 1 precursor Somatostatin precursor Splice isoform 1 of basigin precursor Splice isoform 1 of receptor-type tyrosine-protein phosphatase N2 precursor Splice isoform 3 of integrin alpha-7 precursor

IPI00473056 IPI00299024 IPI00002925 IPI00478414 IPI00395783 IPI00376343 IPI00220562 IPI00219703 IPI00073777 IPI00455739 IPI00000828 IPI00332887 IPI00000130 IPI00218019 IPI00334666 IPI00220749

0.91 0.39 0.89 0.98 0.97 1.04 0.46 1.10 0.67 0.47 0.97 1.00 0.89 0.98 0.92 1.13

NC ↓↓ NC NC NC NC ↓↓ NC ↓↓ ↓↓ NC NC NC NC NC NC

0.93 0.92 1.07 1.31 0.87 1.00 0.99 1.14 0.84 1.03 1.12 1.52 1.10 1.21 0.91 0.84

NC NC NC ↑ NC NC NC NC NC NC NC ↑↑ NC ↑ NC NC

0.63 1.34 1.74 0.55 1.59 0.72 1.43 1.56 1.26 1.79 0.62 0.71 0.56 0.67 0.54 1.51

↓↓ ↑ ↑↑ ↓↓ ↑↑ ↓ ↑ ↑↑ ↑ ↑↑ ↓↓ ↓ ↓↓ ↓↓ ↓↓ ↑↑

Cell Cycle/Death Fas apoptotic inhibitory molecule 2 Hypothetical protein FLJ16490 Latent transforming growth factor beta binding protein 2 Splice isoform 1 of SWI/SNF-related, matrix associated, actin-dependent regulator

IPI00017569 IPI00465099 IPI00465145 IPI00220119

0.88 1.11 1.71 1.61

NC NC ↑↑ ↑↑

0.85 0.99 1.08 0.96

NC NC NC NC

0.56 ↓↓ 0.63 ↓↓ 0.82 ↓ 0.71 ↓

Cell Structure/Motility/Transport/Traffic 12 kDa protein Apolipoprotein C-II precursor Apolipoprotein C-III precursor Divalent cation tolerant protein CUTA Golgi phosphoprotein 2 Hemopexin precursor KIAA1265 protein KIAA1291 protein Latent transforming growth factor-beta-binding protein 2 precursor MIC2L1 isoform E3’-E4’-E3-E4 Neuronal pentraxin receptor isoform 1 Nucleobindin 1 precursor PREDICTED: dynein, cytoplasmic, heavy polypeptide 2 Protein FAM3C precursor Sortilin 1, preproprotein Tetranectin precursor Transthyretin precursor

IPI00477183 IPI00021856 IPI00021857 IPI00034319 IPI00171411 IPI00022488 IPI00008085 IPI00413206 IPI00292150 IPI00152491 IPI00334238 IPI00295542 IPI00171494 IPI00021923 IPI00383591 IPI00009028 IPI00022432

1.04 1.13 0.89 0.82 2.08 1.02 1.08 1.76 1.03 0.86 0.94 0.86 1.07 1.04 1.54 0.63 1.03

NC NC NC ↓ ↑↑ NC NC ↑↑ NC NC NC NC NC NC ↑↑ ↓↓ NC

0.92 1.00 0.57 0.96 0.89 1.05 0.55 1.14 1.00 0.98 0.99 1.13 0.94 1.07 1.06 0.98 1.16

NC NC ↓↓ NC NC NC ↓↓ NC NC NC NC NC NC NC NC NC NC

0.57 1.57 1.28 2.07 0.77 1.64 1.66 0.70 1.25 0.57 0.64 1.62 1.26 1.71 0.65 1.35 1.51

↓↓ ↑↑ ↑ ↑↑ ↓ ↑↑ ↑↑ ↓ ↑ ↓↓ ↓↓ ↑↑ ↑ ↑↑ ↓↓ ↑ ↑↑

Metabolism 2’-phosphodiesterase Angiotensinogen precursor Apolipoprotein C1

IPI00174390 0.91 NC 0.97 NC 0.45 IPI00032220 0.97 NC 0.96 NC 1.23 IPI00021855 1.18 NC 0.99 NC 0.48

↓↓ ↑ ↓↓


305

Table 3, continued AD 1.16 0.57 0.57 0.96 1.10 0.88 0.85 0.91 1.06 0.89 1.04 0.93 0.58 2.33

iTRAQ Ratios PD DLB NC 0.84 NC 0.65 ↓↓ 1.00 NC 1.32 ↓↓ 1.00 NC 1.32 NC 0.88 NC 3.10 NC 1.19 NC 1.88 NC 1.00 NC 1.75 NC 0.88 NC 0.79 NC 1.06 NC 0.66 NC 1.10 NC 1.40 NC 0.96 NC 0.71 NC 0.85 NC 0.24 NC 0.86 NC 0.65 ↓ 1.06 NC 1.68 ↑↑ 0.94 NC 0.71

Protein Name Coagulation factor V Enolase 2 Hypothetical protein DKFZp686B0286 Lysozyme C precursor N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase Probable endonuclease KIAA0830 precursor Rho-associated protein kinase 1 Similar to peptide N-glycanase homolog Sortilin-related receptor precursor Splice isoform 1 of neuroendocrine protein 7B2 precursor Splice isoform 2 of ectonucleotide pyrophosphatase/phosphodiesterase 2 Sulfatase 2 isoform b precursor Superoxide dismutase 1, soluble Transcription elongation regulator 1

IPI # IPI00419311 IPI00216171 IPI00465248 IPI00019038 IPI00009997 IPI00001952 IPI00022542 IPI00165496 IPI00022608 IPI00008944 IPI00303210 IPI00384856 IPI00218733 IPI00247871

Extracellular Matrix/Cell Adhesion Hypothetical protein FLJ35635 Neural cell adhesion molecule 1, 140 kDa isoform precursor T-Cadherin

IPI00385748 0.92 NC 0.89 NC 0.65 ↓↓ IPI00435020 1.15 NC 1.03 NC 0.61 ↓↓ IPI00024046 0.98 NC 0.85 NC 0.58 ↓↓

Immunity/Defense CD99L2 protein Polymeric-immunoglobulin receptor precursor

IPI00434755 1.11 NC 1.63 ↑↑ 0.42 ↓↓ IPI00004573 1.12 NC 0.15 ↓↓ 1.52 ↑↑

Unknown Hypothetical protein FLJ90835 PREDICTED: FLJ46675 protein PREDICTED: G2 protein PREDICTED: hypothetical protein XP 498788

IPI00301255 IPI00165319 IPI00176482 IPI00456355

0.98 1.17 0.92 1.03

NC NC NC NC

Proteins unique to DLB and identified by single peptide Neuronal Activities/Signal Transduction Activating receptor pilrbeta IPI00186781 0.97 NC Hypothetical protein FLJ13782 IPI00016576 0.85 NC Metabotropic glutamate receptor 3 precursor IPI00478165 0.86 NC PREDICTED: KIAA1337 protein IPI00002283 0.97 NC PREDICTED: similar to 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated IPI00376589 0.84 NC protein) Rho-GTPase activating protein 10 IPI00169307 1.00 NC Splice isoform 2 of ephrin type-A receptor 5 precursor IPI00215945 0.88 NC Splice isoform 2 of metabotropic glutamate receptor 8 precursor IPI00396012 0.93 NC

↓↓ ↑ ↑ ↑↑ ↑↑ ↑↑ ↓ ↓↓ ↑ ↓ ↓↓ ↓↓ ↑↑ ↓

1.10 NC 1.29 ↑ 0.89 NC 0.62 ↓↓ 1.28 ↑ 0.58 ↓↓ 0.97 NC 0.63 ↓↓

0.72 0.95 0.51 0.64 1.53

↓ NC ↓↓ ↓↓ ↑↑

1.62 ↑↑ 0.65 ↓↓ 2.43 ↑↑ 1.49 ↑ 0.62 ↓↓

5.99 ↑↑ 0.48 ↓↓ 0.86 NC 0.51 ↓↓ 0.85 NC 0.65 ↓↓

Cell Structure/Motility/Transport/Traffic Hypothetical protein FLJ32363 Laminin gamma-1 chain precursor MGAT3 protein SH3-domain GRB2-like 1 Splice isoform 1 of transcription factor E2-alpha Splice isoform 2 of sodium/potassium/calcium exchanger 2 precursor

IPI00374273 IPI00298281 IPI00020406 IPI00019169 IPI00013929 IPI00218809

1.09 0.93 1.00 1.18 0.84 1.05

NC NC NC NC NC NC

0.88 1.37 1.02 0.89 0.72 0.92

NC ↑ NC NC ↓ NC

0.58 0.60 0.66 0.65 1.74 0.48

↓↓ ↓↓ ↓↓ ↓↓ ↑↑ ↓↓

Metabolism DNA-directed RNA polymerase I largest subunit Hypothetical protein FLJ90551 Neuroendocrine convertase 2 precursor SCMH1 protein Selenoprotein P precursor Splice isoform 2 of glutaryl-CoA dehydrogenase, mitochondrial precursor Splice isoform 3 of reelin precursor


0.59 0.89 1.14 1.06 1.18 0.94 1.05

↓↓ NC NC NC NC NC NC

0.89 1.10 1.11 1.02 0.97 0.93 0.91

NC NC NC NC NC NC NC

1.32 0.56 1.65 0.64 1.71 0.23 0.65

↑ ↓↓ ↑↑ ↓↓ ↑↑ ↓↓ ↓↓

Extracellular Matrix/Cell Adhesion Dermatopontin precursor KIAA1730 protein LOC374654 protein

IPI00292130 1.02 NC 1.11 NC 0.66 ↓↓ IPI00155199 1.63 ↑↑ 1.18 NC 0.66 ↓↓ IPI00394856 1.00 NC 0.93 NC 0.44 ↓↓

306

F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 3, continued

Protein Name Mammalian ependymin related protein 1 Myosin Profilin 2 isoform Splice isoform 2 of collagen alpha 2(VI) chain precursor

IPI # IPI00259102 IPI00218638 IPI00219468 IPI00220613

iTRAQ Ratios PD DLB NC 0.57 ↓↓ 1.55 NC 1.10 NC 1.56 NC 0.84 NC 1.64 NC 0.93 NC 0.55

↑↑ ↑↑ ↑↑ ↓↓

Immunity/Defense Hypothetical protein

IPI00384931 1.03 NC 1.04 NC 2.48

↑↑

Unknown DKFZp434L142 protein HGS RE408 HRPE773 Hypothetical protein FLJ10650 KARCA1 protein Kelch/ankyrin repeat containing cyclin A1 interacting protein PREDICTED: similar to adrenoleukodystrophy protein (ALDP) Protein C20orf98


↓↓ ↓↓ ↑↑ ↓↓ ↓↓ ↓ ↑↑ ↓↓

AD 1.14 1.13 1.12 0.85

0.86 0.85 0.99 1.09 5.81 5.65 1.17 0.90

NC NC NC NC ↑↑ ↑↑ NC NC

0.85 0.93 0.44 0.88 1.06 1.09 1.17 0.96

NC NC ↓↓ NC NC NC NC NC

0.42 0.55 1.78 0.58 0.57 0.76 1.56 0.54

↑↑: Increase (Ratio of AD, PD or DLB vs. control 1.50). ↓↓: Decrease (Ratio of AD, PD, or DLB vs. control 0.67). ↑: Increase (Ratio of AD, PD or DLB vs. control 1.20 < 1.50). ↓: Decrease (Ratio of AD, PD or DLB vs. control > 0.67 0.83). NC: No change (Ratio of AD, PD or DLB vs. control between 0.83 and 1.20).

unique only to AD, PD, or DLB. For instance, we would exclude a protein marker, e.g., calreticulin precursor (IPI00020599), if it were significantly increased not only in AD vs. controls but also PD vs. controls. It should be noted, however, if a marker, e.g., DJ977L11.1 (IPI00478622), displayed a significant increase in AD vs. controls, but a decrease in PD vs. controls, we would consider this marker not only unique to AD but also to PD. With this approach, we identified 136, 72, and 101 proteins that were uniquely altered with AD, PD and DLB, respectively (Table 3). 3.3. Confirmation of candidate protein markers for each neurological disease As demonstrated by data presented in Table 2, the numbers, as well as the types of proteins identified changed significantly when the database was altered. Given that none of the current databases is complete, it is imperative to confirm candidate protein markers not only for their identifications but also for their quantifications as determined by proteomics with alternative means before extensively pursuing their utilities in clinical diagnosis. Currently, there is no high throughput method available for this purpose, and consequently, we utilized conventional Western blot analysis to achieve this goal. Several criteria were used in selecting candidate proteins for further confirmation, including: 1) proteins had to be identified by more than two unique peptides either in IPI database or CDS database

(data not shown); 2) markers should be unique to each disease, i.e., a marker common to two diseases was not considered; 3) markers with known biological functions were preferred; 4) markers identified by both IPI and Celera database were preferred with exception of those with appealing biological functions; and 5) commercial antibodies needed to be available. With these caveats in mind, 16 antibodies were purchased and tested initially with pooled samples that were also used for proteomic analysis. The antibodies chosen were A1BG, ApoC1, ApoC-III, ApoD, ApoH, Ca/CaMKIIB, ceruloplasmin, chromogranin B, β-fibrinogen, furrin, haptoglobin, semaphorin 7A precursor, SPARC (osteonectin), Cu/Zn-SOD, T-cadherin, and VitD BP. Among the 16 antibodies tested with pooled samples, 8 of them were confirmed not only with respect to their identification, i.e. a distinct band was observed in human CSF at appropriate molecular weight for each marker, but also their quantification, meaning that quantitative changes as determined by Western blot were consistent with proteomic assessment for at least one of the diseases. These markers were ApoC1, ApoH, ceruloplasmin, chromogranin B, β-fibrinogen, haptoglobin, T-cadherin, and VitD BP. Western blot results are shown for β-fibrinogen as an example in Fig. 2(A), where quantification was performed with samples being normalized to the amount of protein as well as to the CSF volume. To calculate the sensitivity and specificity for each marker in their ability to differentiate one disease from


307

A: Pooled CSF samples

AD PD DLB CT AD

Normalized to CSF protein

PD

DLB CT

Normalized to CSF volume

B: Individual CSF samples

AD

PD

DLB

CT

AD

PD

DLB

CT

Fig. 2. Confirmation of β-Fibrinogen with Western blot. Proteomic data indicated that β-fibrinogen increased significantly in AD patients over controls with pooled samples. Western blot was used to confirm these results. Panel A: With standard Western blot protocol, pooled samples were analyzed with an antibody against β-fibrinogen (1:2000) both based on total loading amount (10 μg, i.e. similar to proteomic analysis) or CSF volume (10 μl). Panel B: With identical approach in Panel A, but β-fibrinogen was analyzed again in individual samples. AD: Alzheimer’s disease; PD: Parkinson’s disease; DLB: dementia with Lewy body disease; CT: age-matched controls.

controls or from each other, all eight antibodies were then studied in individual samples. Of note, these samples were saved before pooled samples were generated for proteomic analysis and initial confirmation with Western blot analysis mentioned above. The results on Western blot analysis on individual samples are summarized in Table 4, and an actual gel blot is shown in Fig. 2(B) again for β-fibrinogen as an example with the samples being normalized to the amount of protein loaded. Notably, data shown in Table 4 were obtained initially by correcting the OD value of each band to a pooled sample containing all testing control samples and run on the same gel. Next, the data were transformed to percent of controls for ease of comparison with proteomic data. As seen in the Table 4, Western quantification of each marker correlated with proteomic analysis reasonably well, with exception of chromogranin B and T-cadherin, meaning that the results obtained in pooled samples were not replicated in individual ones when tested with Western blot using these two antibodies. It must be kept in mind, however, relative changes may vary between these two techniques simply because the dynamic range for iTRAQ and Western blotting are different.

3.4. Calculation of sensitivity of each marker at 95% specificity Table 5 summarizes the overall discrimination ability of each marker (its AUC) to classify different diseases and controls, the sensitivity at 95% specificity, and the P values from Wilcoxon sum-rank tests comparing two disease groups. From left to right, the AD column in the table provides summaries of AD versus PD, DLB, controls (CT), and non-AD (all three-control groups combined). The PD column presents results compared to DLB, CT, and non-PD, and the third group compares DLB to CT and non-DLB. It appeared that two markers, i.e. β-fibrinogen and VitD BP, can differentiate AD from controls as well as other diseases with AUC at 78% and 88%, respectively, and 50% to 40% sensitivity at 95% specificity. Both Wilcoxon p values are less than 0.05. Similarly, ApoH and ceruloplasmin appeared to be able to segregate PD from controls and other diseases very well. ApoH has the largest AUC and the smallest P value over the eight markers: AUC = 87%, P value = 0.004, and sensitivity at 95% specificity = 67%. Ceruloplasmin was the next best maker with AUC =

308

F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 4 Measurements of each marker in individual cases

Protein ApoH

ApoC1

Ceruloplasmin

Chromogranin B β-Fibrinogen

Haptoglobin

T-Cadherin

VitD BP

AD 1.29 [0.99] (↑) 1.83 [0.89] (*) 1.07 [1.01] (*) 1.14 [0.97] (↓↓) 1.28 [1.35] (↑↑) 2.40 [2.53] (↑↑) 1.28 [1.14] (*) 1.45 [1.22] (*)

PD 1.07 [0.82] (↓↓) 1.89 [0.92] (*) 0.90 [0.85] (↓↓) 1.20 [1.02] (↑↑) 1.07 [1.13] (*) 1.75 [1.84] (↑) 1.10 [0.98] (*) 1.03 [0.87] (↓↓)

DLB 1.33 [1.02] (*) 1.49 [0.72] (↓↓) 1.00 [0.94] (*) 1.01 [0.86] (*) 1.05 [1.11] (*) 1.16 [1.22] (*) 1.13 [1.01] (↓↓) 0.98 [0.82] (*)

Control 1.30 ± 0.036 2.06 ± 0.49 1.06 ± 0.031 1.18 ± 0.071 0.95 ± 0.035 0.95 ± 0.033 1.12 ± 0.077 1.19 ± 0.082

Values (mean ± SE) for each marker are calculated first by correcting OD of each distinct band with the OD of the same band derived from a pooled sample containing all cases and run on the same gel. Value expressed in [ ] are derived from the raw data divided by the mean of control cases shown in the last column, i.e. expressed as percent of controls. The rationale behind data transformation was to replicate the way that proteomic data were obtained. ↑↑, ↓↓: Proteomic changes greater than 50% as compared to controls; ↑: Proteomic changes greater than 20% but less than 50% as compared to controls; *: Proteomic changes less than 20% as compared to controls.

77%, P value = 0.03 and sensitivity at 95% specificity = 56%. However, none of the eight markers were statistically significant predictors of DLB over control or other diseases. Their Wilcoxon P values ranged from 0.07 to 0.9. This may be explained by the marker’s specificity to AD and PD but may also be a result of the small sample size tested for DLB cases (five for discovery and four for confirmation). The fact that none of the single markers could detect AD, PD or DLB with 100% sensitivity at 95% specificity is expected, simply because all neurodegenerative diseases, including AD, PD, and DLB, are heterogeneous in nature, i.e., subgroups of patients may show different markers. Consequently, we next investigated whether higher sensitivity could be achieved by combining individual markers, and the results, shown in Table 6, appeared to indicate that this was indeed the case. Several obvious conclusions can be drawn from the results presented in Table 6. First, the combination of two markers could achieve a higher sensitivity

than a single marker alone. For instance, at 95% specificity, β-fibrinogen and VitD BP had 50% and 40% sensitivity, respectively, when tested alone in differentiating AD from controls and other diseases; but the sensitivity increased to 100% when the two markers were combined, indicating that these markers may be complementary, and not redundant. This finding may degrade when tested on independent samples, but is encouraging, nonetheless. It is also apparent that a better single marker does not necessarily mean it will perform better when the combination approach is taken. This can be illustrated in PD markers, where both ApoH (67%) and ceruloplasmin (56%) had better sensitivity than chromogranin B (11%) when tested alone; but when ApoH was combined with ceruloplasmin and chromogranin B, respectively, the sensitivity remained the same for ApoH+ceruloplasmin, but improved to 78% for ApoH+chromogranin B. In addition, both p values were now at or lower than 0.05 after two markers were combined, indicating that chromogranin B, not ceruloplasmin, will most likely help ApoH outperform ApoH alone. Furthermore, when VitD BP and ApoC1 were combined, the sensitivity for differentiating DLB from other diseases also increased to 50% at 95% specificity and with p value for VitD BP as 0.04 and for ApoC1 as 0.09. Again, it should be emphasized that the results based on sensitivity and specificity related to DLB should be considered provisional given the limited sample size. Last, but not least, no overt improvement was seen when a third maker was added to composite marker panel (data not shown), which can be expected with the small sample sizes used here. Results on the performance of composite markers (CM) as well as ROC curves for both single and CM are also shown graphically in Figs 3 and 4, where the joint behaviors of standardized markers among disease and control groups are displayed. As clearly shown in Figs 3 and 4, the ability of CM to separate AD or PD from other diseases or healthy controls was better when two dimensions were used instead of one. Similarly, both ROC curve plots showed the improvement of sensitivity over all the ranges of specificity of CM compared to each individual marker. The statistical significance of the logistic regression suggested that both markers were significant and important contributors to the resulting CM ROC curve. 4. Discussion The advances made from current investigations can be summarized as the following: 1) a total of more than


309

Table 5 Summary of ROC curves for eight markers Case Group Control Group ApoC1 AUC Sense (0.95) Wilcoxon P value ApoH AUC Sense (0.95) Wilcoxon P value Chromogranin B AUC Sense (0.95) Wilcoxon P value Ceruloplasmin AUC Sense (0.95) Wilcoxon P value β-Fibrinogen AUC Sense (0.95) Wilcoxon P value Haptoglobin AUC Sense (0.95) Wilcoxon P value T-cadherin AUC Sense (0.95) Wilcoxon P value VitD BP AUC Sense (0.95) Wilcoxon P value

PD

DLB

AD CT

NonAD

DLB

PD CT

0.478 0.000 0.659

0.625 0.100 0.536

0.633 0.000 0.360

0.536 0.000 0.984

0.667 0.222 0.412

0.806 0.600 0.038*

0.550 0.400 0.835

0.525 0.300 0.896

0.610 0.100 0.329

0.611 0.111 0.438

0.750 0.556 0.214

0.574 0.000 0.603

0.772 0.444 0.811

0.708 0.444 0.336

0.733 0.500 0.111

DLB NonPD

CT

NonDLB

0.605 0.000 0.489

0.495 0.000 0.740

0.667 0.000 0.412

0.656 0.250 0.354

0.861 0.778 0.078

0.944 0.778 0.008*

0.869 0.667 0.004*

0.391 0.250 0.461

0.593 0.250 0.580

0.525 0.111 0.779

0.847 0.667 0.101

0.525 0.000 0.862

0.621 0.111 0.315

0.806 0.000 0.131

0.801 0.000 0.073

0.457 0.111 0.728

0.672 0.444 0.168

0.667 0.556 0.413

0.809 0.556 0.041*

0.765 0.556 0.030*

0.722 0.000 0.270

0.440 0.000 0.620

0.725 0.500 0.251

0.844 0.600 0.023*

0.777 0.500 0.020*

0.556 0.333 0.821

0.756 0.444 0.095

0.493 0.000 0.967

0.722 0.250 0.270

0.531 0.000 0.888

0.594 0.300 0.496

0.675 0.500 0.375

0.811 0.600 0.038*

0.702 0.300 0.087*

0.667 0.333 0.413

0.815 0.667 0.041

0.600 0.000 0.364

0.722 0.250 0.270

0.545 0.000 0.800

0.622 0.200 0.402

0.625 0.300 0.535

0.589 0.400 0.548

0.609 0.300 0.347

0.556 0.222 0.821

0.549 0.111 0.794

0.578 0.000 0.507

0.500 0.250 0.940

0.554 0.000 0.932

0.950 0.800 0.005

0.975 0.900 0.021*

0.767 0.400 0.071

0.880 0.400 0.002*

0.555 0.222 0.821

0.685 0.222 0.233

0.758 0.222 0.037*

0.778 0.000 0.168

0.777 0.000 0.092

AUC denotes the area under ROC curve whereas Sense (0.95) denotes the sensitivity at 95% specificity of the ROC curve. The Wilcoxon sum-rank test with P value less than 0.05 was marked by an asterisk. Table 6 Summary for composite markers

AD versus all others PD versus all others DLB versus all others

Marker 1

Marker 2

AUC

VitD BP Ceruloplasmin ApoH Ceruloplasmin ApoC1 ApoC1

β−Fibrinogen β−Fibrinogen Chromogranin B Chromogranin B Chromogranin B VitD BP

0.99 0.94 0.92 0.93 0.92 0.86

Sense (0.95) 1.00 0.89 0.78 0.56 0.50 0.50

P-value for marker 1 0.0635 0.0142 0.0086 0.0052 0.0737 0.0394

P-value for marker 2 0.0207 0.0199 0.0560 0.0203 0.086 0.0890

AUC denotes the area under ROC curve whereas Sense (0.95) represents the sensitivity at 95% specificity of the ROC curve for CM of marker 1 and 2. P value for marker 1 represents the likelihood ratio P value from logistic regression for marker 1 given marker 2, whereas P value for marker 2 represents the P value of marker 2 given marker 1 in the logistic regression model.

1,500 proteins were identified and quantified in wellcharacterized pooled human CSF; 2) among markers that were altered by disease states, those unique to AD, PD and DLB were discovered; 3) eight unique markers as determined by quantitative proteomics were further

confirmed by Western blot analysis for their quantification as well as identification in individual samples; and 4) preliminary analysis of these markers in individual samples suggested that when more than one marker was used, AD, PD, and potentially DLB could be separated

310


B 1.0

A

0.8 0.6 0.2

0.4

Sensitivity

4 2 0

CM VitD BP Beta-Fibrinogen

0.0

-2

Standardized Beta-Fibrinogen

6

AD PD DLB Control

-3

-2

-1 0 1 Standardized VitD BP

2

3

0.0

0.2

0.4 0.6 1-Specificity

0.8

1.0

Fig. 3. Composite markers for AD vs. others. Panel A: The scatter plot displays the association of standardized β-fibrinogen and standardized VitD BP with AD, PD, and DLB cases and healthy controls. Line in plot represents the composite marker defined from logistic regression. The actual line represented here gives the classification rule for 95% specificity. Panel B: ROC curves for VitD BP, β-fibrinogen, and the composite marker (CM). The following statistics are obtained from CM: AUC (area under curve) = 0.99; Sensitivity at 95% specificity = 1.00 with p-value for VitD BP = 0.0635 and p-value for β-fibrinogen = 0.0207.

A

0.8 0.6 0.2

0.4

Sensitivity

-1

0

-6

-4

-2 0 Standardized ApoH

2

4

CM ApoH ChromograninB

0.0

1

PD AD DLB Control

-2

Standardized ChromograninB

2

1.0

B

0.0

0.2

0.4 0.6 1-Specificity

0.8

1.0

Fig. 4. Composite markers for PD vs. others. Panel A: The scatter plot displays the association of standardized chromogranin B and standardized ApoH with AD, PD and DLB cases and healthy controls. Line in plot represents the composite marker defined from the logistic regression. The actual line represented here gives the classification rule for 95% specificity. Panel B: ROC curves for chromogranin B, ApoH and the composite marker (CM). The following statistics are obtained from CM: AUC (area under curve) = 0.92; Sensitivity at 95% specificity = 0.78 with p-value for chromogranin B = 0.0056 and p-value for ApoH = 0.0068.

from controls as well as from other diseases with high sensitivity at 95% specificity. We have recently identified close to 1,000 proteins in the CSF of healthy young adults [52], and some of which appear to change with aging [54] and in patients with AD [55]. Compared with our previous results,

1,090 new proteins were identified, expanding the total CSF proteome to 1,882 proteins, the most extensive characterization of human CSF proteins to date. The significant increase in the number of proteins identification in this study largely resulted from two major factors: 1) better separation of peptides by extensive


chromatography; and 2) utilization of a more advanced MS instrument. Extensive chromatography is essential in LC based proteomics, even when MudPIT is used, as complex samples usually yield hundreds of thousands of peptides after proteins are digested. This issue is especially challenging in proteomic analysis of CSF or plasma, where albumin and IgGs constitute more than 75% of total proteins [5]. Here, we achieved extensive peptide separation by utilizing two consecutive processes: 1) perform RP separation with a nano-capillary LC system that increases sensitivity by at least ten folds as compared to a conventional micro-capillary LC system, resulting in less peptides eluted onto each MALDI plate; and 2) spot each LC run to 24 × 24 (576) arrays on an MALDI plate instead of a standard 198 spot array, thereby further separating peptides. Good peptide separation is evidenced by the fact that more than 400 (417 to be precise) proteins were identified in 1.5P fraction, notwithstanding it was overtly enriched in albumin and IgGs [54]. The significance of extensive identification of the human CSF proteome is apparent, as it not only substantially expands our current knowledge regarding human CSF proteins, but also provides information needed to appropriately interpret protein biomarkers of agerelated neurodegenerative diseases. In addition, the impact of this data will likely reach beyond neurodegenerative diseases because intense interest has also been expressed in other CNS diseases, including multiple sclerosis [17], acute brain injury [45], and CNS tumors [56]. Many candidate markers were discovered for AD patients in this study. As many groups, including us, have investigated AD CSF markers in the past with proteomic approaches [4,6,7,40,55], one should wonder how our current results compare with those reported in the literature, or at the very least, to our own results. While comparison to our own results is valid and meaningful, it is very hard, if not impossible, to make a fair comparison between our results with those of other groups. This is because there are many variables involved in proteomic studies, including difference in sample preparation, quality control of CSF samples (particularly potential blood contamination), patient population, proteomic platforms used, criteria used for protein identification, whether identified proteins have been confirmed or validated, and type of database used, which is a critical issue as demonstrated in this and our previous studies [52]. Nonetheless, we have compiled all of the results generated by all platforms of proteomics in Table 7, demonstrating, as

311

expected, that only a very small fraction of proteins change in the same direction, whether increase or decrease in AD vs. controls, among all or most studies. The increased proteins are albumin precursor, amyloid beta A4 protein precursor, α-1-antitrypsin precursor, ApoA-II precursor, complement C4 precursor, a hypothetical protein (IPI00300241), β-2-microglobulin (isoforms) (with one exception), neuronal pentraxin I precursor, retinol binding protein (with one exception), and thioredoxin (except in our previous study where this protein was not quantified). There are only two consistently decreased proteins, i.e., β-1,3N-acetylglucosaminyltransferase bGnT-6 and EWI2, when AD patients are compared to controls. The concordance between our previous and current studies is higher, as in addition to the proteins mentioned above, the following proteins also demonstrate similar quantitative changes: afamin precursor, chemokine (C-X-C motif) ligand 16, β-galactosidase binding lectin precursor, GM2 activator precursor, ganglioside, α-2-macroglobulin precursor, and seleniumbinding protein 1. Finally, 6 proteins displayed significant changes in the current study (Table 3) in the same direction, whether increased or decreased in AD vs. controls, as those listed in our previous publication where they have changes > 20% but < 50% alternations [55], and consequently are not listed in Table 7. These proteins are: dystroglycan precursor, haptoglobin, hemopexin precursor, ribonuclease 6 precursor, mimecan precursor, and tetranectin precursor. What is most remarkable is that among all of the proteins with consistent changes in most experiments, only very few are unique to AD, i.e. most previous “candidate” markers were also changing more or less in the same direction in PD or DLB cases. Because it is not difficult for an experienced clinician to diagnose demented subjects from controls, the utility of these “non-unique” markers diminishes significantly. The only unique marker that has been consistently found in all studies is ApoA-II precursor. The other close possibility is haptoglobin, which is why we selected this protein for further confirmation even though it also displayed more than 20% changes in PD vs. controls. These results emphasize again that it is imperative to include other disease controls in addition to age-matched controls when the goal is to identify unique disease markers. Candidate markers unique to AD fall into all six major categories: neuronal activities/signal transduction, cell cycle/death, cell structure/motility/transport/traffic, metabolism, extracellular matrix/cell adhesion, and im-

312

F. Abdi et al. / Detection of biomarkers with a multiplex quantitative proteomic platform Table 7 Comparison of AD CSF markers across all proteomic studies No. 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

28 29 30

31 32 33 34 35 36 37 38 39 40 41 42 43

Name (IPI) IPI00004656 IPI00182398 IPI00032220 * IPI00022434 IPI00305457 IPI00216722 IPI00022431 IPI00006608 IPI00219182 IPI00219189 IPI00021841 IPI00479805 IPI00021842 NCBI 178855* IPI00021854 GenBank ID 4557327* IPI00009997 IPI00181232 IPI00234495 IPI00020984 IPI00234495 IPI00257600 NCBI 4758048 * GenBank ID 4557018* IPI00032258 IPI00233778 IPI00032293 IPI00027547 IPI00033086 IPI00221115 IPI00221116 IPI00056478 IPI00186736 IPI00022371 IPI00183616 IPI00218719 IPI00233252 IPI00300241 IPI00333982 IPI00168728 IPI00045498 IPI00011274 IPI00332161 IPI00328111 IPI00245370 IPI00170706 IPI00215894 IPI00025456 IPI00155723 IPI00215638 IPI00027381 IPI00064607 IPI00032292 IPI00013299

Previous study [54,55] ↑↑

Peer Literature NI

Current study NI

↑↑ NI ↑ ↑↑ NC ↑ ↑↑

NI ↑ [6] ↓ [40] ↑ [40] ↓ [19,40] ↓ [40] NI

NC NI ↑↑ ↑ NI NI ↑

Identified NI Identified Identified ↑↑ NI

↓ [40] ↓ [10] ↓ [10,40] ↓ [40] NI ↓ [19]

NI ↑↑ NI NI ↑↑ NI

Beta-1,3-N-acetylglucosaminyltransferase bGnT-6 Ca2+ -dependent activator protein for secretion 2 Cathepsin B preproprotein Calnexin Cathepsin B preproprotein Cell adhesion molecule with homology to L1CAM precursor Cell cycle progression 8 protein Chitinase 3-like 1

↓↓ ↑↑ ↑↑ ↓↓ ↑↑ ↓↓ NI NI

NI NI NI NI NI NI ↓ [40] ↑ [19]

↓ NI NI NI NI NI NI NI

Complement C4 precursor Complement component 1, r subcomponent Cystatin C Dermcidin precursor Disks large-associated protein 2 Splice isoform 2 of Q9P1A6 Disks large-associated protein 2 Splice isoform 3 of Q9P1A6 Disks large-associated protein 2 EWI2 LIR-D1 Histidine-rich glycoprotein precursor Hypothetic protein Splice isoform 2 of P78527 DNA-dependent protein kinase catalytic subunit DNA-dependent protein kinase catalytic subunit Hypothetical protein Hypothetical protein FLJ00385 protein Hypothetical protein, JKTBP1delta6 Heterogeneous nuclear ribonucleoprotein D-like Ig gamma-1 chain C region Factor VII active site mutant immunoconjugate Insulin-like growth factor binding protein 2 (36 kD) KIAA1412 protein Kininogen precursor LJ00053 protein Leukophysin DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9 isoform 1 Lymphocyte antigen 75 precursor MEGF10 protein Metalloproteinase inhibitor 1 precursor Neuroblastoma, suppression of tumorigenicity 1

↑↑ ↑↑

NI NI

↑ NI

NC ↓↓ ↓↓

↑ [6,19] NI NI

NC ↑↑ NI

↓↓ ↓↓ ↑↑ ↓↓

NI NI NI NI

↓ NC NC NI

↑↑ ↑↑

NI NI

↑↑ NI

↓↓

NI

NI

↑↑

NI

NI

↑↑ ↓↓ Identified ↓↓ ↓↓

NI NI ↓ [40] NI NI

NI NI NI NI NI

↓↓ ↑↑ ↓↓ ↑↑

NI NI NI NI

NI NI NC NI

Common Name Alpha-2-microglobulin precursor Hypothetic protein Angiotensinogen precursor 7.7 kDa unknown protein Albumin precursor Alpha-1-antitrypsin precursor Alpha-1β glycoprotein Alpha-2-HS glycoprotein Amyloid beta A4 protein precursor

ApoA1 ApoA4 ApoE ApoJ Apolipoprotein A-II precursor # Apolipoprotein H precursor


313

Table 7, continued Comparison of AD CSF markers across all proteomic studies No. 44 45 46 47 48 49

50 51 52 53 54

55 56 57 58 59 60 61

Name (IPI) IPI00220562 IPI00027425 IPI00022284 IPI00170548 178775* IPI00001508 NCBI 730305; GenBankID 455962672* IPI00479848 IPI00244477 IPI00232736 IPI00171473 IPI00219020 IPI00177441 IPI00180675 IPI00179709 IPI00183040 IPI00166768 IPI00216005 IPI00218345 IPI00216298 IPI00022463 IPI00022432 IPI00383014 IPI00298853 4699583* IPI00004656

Previous study [54,55] ↑↑ ↓↓

Peer Literature NI NI

Current study ↑ NC

PRO2000 protein Proapolipoprotein Proinsulin precursor Protaglandin D2 synthase

↓↓ NI ↑↑ NC

NI ↓ [10] NI ↓ [40] ↑ [19]

NI NI NI NI

Retinol binding protein Similar to fem-1 homolog a Similar to RIKEN cDNA 2410146L05 Spondin 1, (f-spondin) extracellular matrix protein a. Splice isoform 1 of Q13748 tubulin alpha-2 chain b. Similar to tubulin alpha-3/alpha-7 chain

↑ ↑↑ ↓↓ ↑↑ ↑↑

↓ [40] ↑ [10] NI NI NI NI

↑ NI NI NC NI

NI ↑ ↑ Identified ↓↓ NI ↑↑

↑ [19] ↓ [40] ↓ [40] ↑ [10] ↓ [6] NI ↑ [10] ↑ [6,10,19] ↓ [40]

↑ NI NC ↑ NC NI ↑

Common Name Neuronal pentraxin I precursor Prion protein

c. Hypothetical protein

Tubulin alpha-8 chain Tubulin, alpha 2 isoform 2 Thioredoxin Transferrin precursor Transthyretin VGF protein Vitamin D-binding protein precursor Zn-α-2 glycoprotein β-2-Microglobulin (isoforms)

↑↑: Increase (Ratio of AD vs. control 1.50). ↓↓: Decrease (Ratio of AD vs. control 0.67). ↑: Increase (Ratio of AD vs. control 1.2 < 1.50 in our studies, but only denotes trend of increase in other proteomic experiments). ↓: Decrease (Ratio of AD vs. control > 0.67 0.83 in our studies, but only denotes trend of decrease in other proteomic experiments). NC: No change (Ratio of AD vs. control between 0.83 ∼ 1.20). NI: Not identified. *: No IPI number as they are identified by others using different database. #: Protein unique to AD as listed on Table 3. Multiple International Protein Index (IPI) entries in a box, e.g. Box 8, signify one protein with multiple IPI in the database. Multiple entires designated by lowcase letters in a box before common names, e.g. Box 54, indicate multiple possible candidates from the sequenced peptides, tytically isoforms and precursors. Both were determined in our previous studies with ProteinProphet analysis [54, 55,52]. As ProteinProphet, generated initially for SEQUEST based protein identification, has not been able to interface with MASCOT based search to date, all protein identification in the current study (derived from MASCOT) was based on unique IPI.

munity/defense (Table 3). It is not practical to discuss each of the candidate proteins in detail; thus, our discussion will be focused on three confirmed markers demonstrating relatively high sensitivity, i.e. βfibrinogen, haptoglobin, and VitD BP. β-Fibrinogen, best known for its role in coagulation and inflammation, has at least two isoforms, α and β, and it is not clear whether this protein is synthesized in the brain or transported via the blood brain barrier (BBB) [47]. Nonetheless, it has been recognized for some time now that the activity of fibrinogen is increased in the plasma of AD patients [16]. The role of increased β-fibrinogen in AD CSF is not clear, but it can be at least hypothe-

sized that it could potentially enhance microglial activation, a process implicated as one of the major mechanisms of cell death in AD [44]. On the other hand, an increase in haptoglobin in CSF has been associated with a subpopulation of AD patients, and it is initially [39] thought to be due to an abnormal penetration of haptoglobin in AD patients secondary to compromised BBB [1,49]. Some studies have also associated haptoglobin with increased risk in some AD patients, although contradictory results have also been reported [31]. Nonetheless, several studies performed with conventional methods also show that the level of haptoglobin increases in AD patients [23]. Conversely, as

314


demonstrated in this study, an increase in haptoglobin alone is not sufficient to differentiate AD from other neurological diseases [23]. VitD BP has been classically associated with calcium metabolism and bone remodeling, although recently it has been noted that mRNA levels for this protein are decreased in the hippocampus in Alzheimer’s patients [48]. Nevertheless, like haptoglobin, the role of VitD BP in the pathogenesis of AD is largely unknown. Prior to this study, very little was known about markers unique to PD and DLB. Three confirmed good candidate markers for PD are ceruloplasmin, chromogranin B and ApoH. Ceruloplasmin is an interesting protein because it has been implicated to play a central role in PD pathogenesis owing to two observations: 1) iron deposition in PD substantia nigra correlates with the severity of the disease [18]; and 2) ceruloplasmin, an important protein for iron transportation, is decreased in the blood of PD patients [50]. Finally, It should be emphasized that this protein did not decrease in all PD patients in an earlier study [29], consistent with the facts that this disease is heterogeneous in nature and that it is probably not sufficient by itself to detect PD patients with high sensitivity at high specificity. The influence of chromogranin B, a non-significant marker by itself in confirmation studies, on the overall performance of ApoH is also remarkable, as it significantly improved the sensitivity of ApoH to differentiate PD from controls as well as other diseases. Notably, a study performed years ago has suggested that although chromogranin B cannot differentiate AD or PD from controls by itself, the ratio between chromogranin A and B may be a correcting factor for neuropeptides seen in human CSF [11]. The third marker that might be important in PD is ApoH, a protein clearly reported to be present in human CSF [26], although its role in PD or in neurodegenerative diseases in general remains to be defined. The markers unique to DLB also appeared to be related to two lipoproteins, i.e., ApoC1 and ApoH. Again, the role of ApoH in DLB or neurodegenerative disease in general is largely unknown, although given the fact that it also increased in PD, one might argue its role in Lewy body disease. Very little is known about the role of ApoC1 in Lewy body disease, including PD and DLB, or dementia. However, several issues are worth commenting. First, one does not need to know the function of a protein in order for it to be a diagnostic tool; a good example of this type of use is the presence of oligoclonal bands in the CSF in the absence of identical bands in serum, which has been widely used clinically

to aide the diagnosis of multiple sclerosis [36]. Second, all of these novel proteins should be studied further not only for their diagnostic use, but also for their roles in the pathogenesis of neurodegenerative diseases. Finally, given the limited DLB cases studied, caution needs to be excised with respect to the significance of these proteins. Another significant issue that needs to be discussed relates to the fact that not all proteins discovered by proteomics were confirmed by Western blot analysis with respect to their relative quantification. Given the caveats associated with incompleteness of current databases, one could argue that a protein confirmed with Western blot may not be the protein quantified by the proteomics. Another reason for poor correlation of Western results with proteomic analysis in some protein markers may result from the possibility that the amino acids labeled by iTRAQ may be partially modified in some peptides, thereby influencing the average ratio of quantification which translated into quantitative changes as determined by proteomics, but showed no changes when assessed by Western blot. Lastly, the dynamic range of a Western is not as good as mass spectrometry, i.e., a 20% increase as determined by Western blot does not mean necessary that it is different from a 50% increased as assessed by proteomic data. However, despite all the caveats discussed, the fact remains that a fraction of proteins identified by proteomic profiling were indeed confirmed by alternative means, and several markers are very promising in segregating AD, PD, and DLB from controls as well as from each other. Examples of potentially good combination of markers included β-fibrinogen plus VitD BP or ceruloplasmin for AD, chromogranin B plus ceruloplasmin or ApoH for PD and ApoC1 plus chromogranin B or VitD BP for DLB (Table 6). In summary, we have identified 1,090 new proteins in the current study, thereby expanding the human CSF proteome to 1,882 proteins. Of these, 136, 72, and 101 proteins displayed changes unique to AD, PD and DLB, respectively. Some of these candidate proteins were further confirmed with Western blot with pooled sample first, followed by confirmation in individual samples. Finally, sensitivity at 95% specificity of each marker was calculated alone and in combination in its ability to differentiate AD, PD, and DLB from controls as well as from each other. It should be emphasized that these results, though encouraging, need to be validated in a larger and different population of patients, which are currently being collected at our institutions as well as in collaboration with other centers.


Acknowledgements Proteomic characterization of human CSF is supported by grants to JQ (NIH: AG08017; Dept of Veteran’s Affairs Career Development Award; and the Dana Foundation), to TJM (NIH: AG05136 and NS048595) and to JZ (NIH: AG025327 and ES012703 as well as the Michael J. Fox Foundation). We also deeply appreciate those who have donated their CSF for our studies.

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317

Appendix I (Proteins identified by 2 or more peptides)

Protein Name

IPI Address

10 kDa protein 10 kDa protein 107 kDa protein 11 kDa protein 12 kDa protein 132 kDa protein 132 kDa protein 154 kDa protein 16 kDa protein 19 kDa protein 2,3-bisphosphoglycerate mutase 21 kDa protein 24 kDa protein 25 kDa protein 29 kDa protein 2’-phosphodiesterase 38 kDa protein 39S ribosomal protein L9, mitochondrial precursor 42 kDa protein 44 kDa protein 45 kDa calcium-binding protein precursor 45 kDa protein 54 kDa protein 57 kDa protein 59 kDa protein 59 kDa protein 61 kDa protein 65 kDa protein 72 kDa type IV collagenase precursor 75 kDa protein 75 kDa protein 85 kDa protein 9 kDa protein Actin, alpha skeletal muscle Actin, cytoplasmic 1 Actin, cytoplasmic 2 Adenovirus E3-14.7K interacting protein 1 Adenylate cyclase type III Adenylate cyclase, type IX Adseverin AF5q31 protein Afamin precursor Agrin ALB protein

IPI00477452 IPI00478205 IPI00476999 IPI00479928 IPI00477183 IPI00412845 IPI00477893 IPI00472011 IPI00328348 IPI00181341 IPI00215979 IPI00477336 IPI00479531 IPI00334282 IPI00180776 IPI00174390 IPI00333662 IPI00307409 IPI00333429 IPI00479267 IPI00106646 IPI00478761 IPI00473015 IPI00479902 IPI00479340 IPI00479977 IPI00334408 IPI00479169 IPI00027780 IPI00238755 IPI00413996 IPI00414205 IPI00477785 IPI00021428 IPI00021439 IPI00021440 IPI00105620 IPI00028513 IPI00030099 IPI00002606 IPI00004344 IPI00019943 IPI00479925 IPI00384697

318


Protein Name

IPI Address

Alcadein alpha-1 ALDOC protein Alpha 2,6-sialyltransferase AlphA 3 type VI collagen isoform 3 precursor Alpha-1,3 Alpha-1-acid glycoprotein 2 precursor Alpha-1-antitrypsin precursor Alpha-1B-glycoprotein precursor Alpha-2-antiplasmin precursor Alpha-2-glycoprotein 1, zinc Alpha-2-HS-glycoprotein precursor Alpha-2-macroglobulin precursor Alpha-2-macroglobulin receptor-associated protein precursor Alu subfamily SB sequence contamination warning entry AMBP protein precursor Amyloid-like protein 1 precursor Angiotensinogen precursor Antithrombin III variant ApoA4 protein Apobec-1 stimulating protein Apolipoprotein A-I precursor Apolipoprotein A-II precursor Apolipoprotein A-IV precursor Apolipoprotein C-I precursor Apolipoprotein C-II precursor Apolipoprotein C-III precursor Apolipoprotein D precursor Apolipoprotein E precursor Apolipoprotein M Associated molecule with the SH3 domain of STAM ATP-binding cassette, sub-family A member 8 ATP-binding cassette, sub-family A, member 2 isoform b Baculoviral IAP repeat-containing protein 1 Basement membrane-specific heparan sulfate proteoglycan core protein precursor Beta galactosyltransferase Beta-2-glycoprotein I precursor Beta-2-microglobulin precursor Beta-galactosidase binding lectin precursor Beta-globin gene from a thalassemia patient, complete cds Betaglycan Biotinidase precursor BK134P22.1 Bone morphogenetic protein 3b precursor Bone-derived growth factor Brain abundant, membrane attached signal protein 1 Brain immunoglobulin receptor precursor Brain-derived neurotrophic factor BDNF1 Butyrophilin C1orf16 protein

IPI00007257 IPI00418262 IPI00479942 IPI00072917 IPI00061448 IPI00020091 IPI00305457 IPI00022895 IPI00029863 IPI00166729 IPI00022431 IPI00478003 IPI00026848 IPI00383860 IPI00022426 IPI00020012 IPI00032220 IPI00032179 IPI00479805 IPI00299499 IPI00021841 IPI00021854 IPI00304273 IPI00021855 IPI00021856 IPI00021857 IPI00006662 IPI00021842 IPI00030739 IPI00290975 IPI00479296 IPI00414303 IPI00011547 IPI00024284 IPI00184094 IPI00298828 IPI00004656 IPI00219219 IPI00382950 IPI00304865 IPI00218413 IPI00009619 IPI00023315 IPI00015916 IPI00299024 IPI00166048 IPI00336003 IPI00384734 IPI00448672


319

Protein Name

IPI Address

C4B1 CAD protein Cadherin EGF LAG seven-pass G-type receptor 2 precursor Cadherin-13 precursor Calcium binding protein Calmodulin Calmodulin-like 3 CAP-binding protein complex interacting protein 1 isoform A Carboxypeptidase E precursor Cathepsin B precursor Cathepsin D precursor CD59 glycoprotein precursor CD99L2 protein Cell growth regulator with EF hand domain 1 Cell growth regulator with EF hand domain 1 Ceruloplasmin precursor Chitinase-3 like protein 1 precursor Cholecystokinins precursor Chordin-like 1 Chromogranin A Chromogranin A precursor Ciliary dynein heavy chain 9 Ciliary rootlet coiled-coil, rootletin Clusterin isoform 1 Clusterin precursor Coagulation factor V Cocaine- and amphetamine-regulated transcript protein precursor Cochlin precursor Collagen alpha 1(I) chain precursor Collagen alpha 1(VI) chain precursor Collagen alpha 2(I) chain precursor Collagen alpha 2(V) chain precursor Complement C1r subcomponent precursor Complement C1s subcomponent precursor Complement C2 precursor Complement C3 precursor Complement C4 precursor Complement C5 precursor Complement component 4B proprotein Complement component C6 precursor Complement component C7 precursor Complement component C8 beta chain precursor Complement component C8 gamma chain precursor Complement component C9 precursor Complement factor I precursor Contactin 2 precursor COP9 signalosome complex subunit 4 Corticosteroid-binding globulin precursor C-type natriuretic peptide precursor


320


Protein Name

IPI Address

Cyclophilin Cystatin C precursor Cytochrome P450 27, mitochondrial precursor Cytokeratin type II Cytosolic malate dehydrogenase D1 dopamine receptor-interacting protein calcyon DA141H5.1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 51 Death-associated protein kinase 1 Dickkopf related protein-3 precursor Dihydropyridine-sensitive L-type, calcium channel alpha-2/delta subunits precursor Divalent cation tolerant protein CUTA DJ1071L10.1 DPKL1915 Dystroglycan precursor Endothelin B receptor-like protein-2 precursor Enolase 2 Ephrin type-A receptor 4 precursor Ephrin-B2 precursor Epididymal secretory protein E1 precursor Esophageal cancer related gene 4 protein EWI2 Exostosin-like 2 Extracellular matrix protein 1 Extracellular matrix protein 1 precursor Extracellular sulfatase sulf-2 precursor Extracellular superoxide dismutase [Cu-Zn] precursor Far upstream element binding protein 2 F-box only protein 10 Fc fragment of IgG binding protein Fibrinogen beta chain precursor FK506-binding protein 1A FLJ00120 protein FLJ00268 protein FLJ00271 protein FLJ00412 protein FLJ35220 protein Follistatin-like 4 Full-length cDNA 5-prime end of clone CS0DM009YC13 of fetal liver of homo sapiens Full-length cDNA clone CS0DC025YL05 of neuroblastoma of homo sapiens Full-length cDNA clone CS0DH002YN05 of T cells Full-length cDNA clone CS0DI028YM15 of placenta of homo sapiens Full-length cDNA clone CS0DI085YI08 of placenta of homo sapiens Full-length cDNA clone CS0DN001YP04 of adult brain of homo sapiens Furin precursor G protein coupled receptor 158 Galectin-3 binding protein precursor Ganglioside GM2 activator precursor GARS protein



321

Protein Name

IPI Address

GBP protein isoform A Gelsolin isoform B Glial fibrillary acidic protein, astrocyte Glucosidase II beta subunit precursor Glutamate receptor 4 precursor Gm133 GM2 activator protein Golgi autoantigen, golgin subfamily B member 1 Golgi phosphoprotein 2 Golgin-67 isoform C Growth/differentiation factor 11 precursor Growth/differentiation factor 8 precursor Growth-arrest-specific protein 2 Haptoglobin precursor Hect domain and RLD 4 Hemoglobin alpha-1 globin chain Hemoglobin gamma-G Hemopexin precursor Heparin-binding EGF-like growth factor precursor HERC2 protein HGFL(S) protein Histidine-rich glycoprotein precursor Histone deacetylase 11 HLA class I histocompatibility antigen, alpha chain H precursor HLA class I histocompatibility antigen, B-27 alpha chain precursor HLA class I histocompatibility antigen, B-35 alpha chain precursor HLA class I histocompatibility antigen, B-54 alpha chain precursor HLA class I histocompatibility antigen, B-67 alpha chain precursor HLA class I histocompatibility antigen, Cw-15 alpha chain precursor HLA class I histocompatibility antigen, Cw-2 alpha chain precursor HLA class I histocompatibility antigen, Cw-7 alpha chain precursor HLA class I histocompatibility antigen, E alpha chain precursor HLA-C protein Hook homolog 3 HSAJ1454 HU-K4 Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein


322


Protein Name

IPI Address

Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein DKFZp451B1418 Hypothetical protein DKFZp566O224 Hypothetical protein DKFZp686B0286 Hypothetical protein DKFZp686C02220 Hypothetical protein DKFZp686C15213 Hypothetical protein DKFZp686E04229 Hypothetical protein DKFZp686I15212 Hypothetical protein DKFZp686J1375 Hypothetical protein DKFZp686K11107 Hypothetical protein DKFZp686L19235 Hypothetical protein DKFZp686P15220 Hypothetical protein DKFZp761H2024 Hypothetical protein DKFZp761O0610 Hypothetical protein FLJ14473 Hypothetical protein FLJ16025 Hypothetical protein FLJ16420 Hypothetical protein FLJ16490 Hypothetical protein FLJ16561 Hypothetical protein FLJ20421 Hypothetical protein FLJ23121 Hypothetical protein FLJ25359 Hypothetical protein FLJ25530 Hypothetical protein FLJ31726 Hypothetical protein FLJ33516 Hypothetical protein FLJ33620 Hypothetical protein FLJ33674 Hypothetical protein FLJ35588 Hypothetical protein FLJ35635 Hypothetical protein FLJ42206 Hypothetical protein FLJ43748 Hypothetical protein FLJ43983 Hypothetical protein FLJ44823 Hypothetical protein FLJ46033 Hypothetical protein FLJ46113 Hypothetical protein FLJ90018 Hypothetical protein FLJ90651 Hypothetical protein FLJ90761 Hypothetical protein FLJ90835 Hypothetical protein GS103



323

Protein Name

IPI Address

Hypothetical protein MOT8 Hypothetical protein PSEC0072 Hypothetical protein PSEC0164 Hypothetical protein SGCE Hypothetical protein WUGSC:H NH0436C12.1 Ig heavy chain V-I region HG3 precursor Ig heavy chain V-I region V35 precursor Ig heavy chain V-III region CAM Ig kappa chain V-I region AG Ig kappa chain V-I region DEE Ig kappa chain V-I region EU Ig kappa chain V-I region Ni Ig kappa chain V-I region OU Ig kappa chain V-III region B6 Ig kappa chain V-III region WOL Ig lambda chain V-I region WAH IGHG1 protein IGHG1 protein IGHG4 protein IGHM protein Insulin-like growth factor binding protein 2 precursor Insulin-like growth factor binding protein 4 precursor Insulin-like growth factor binding protein 5 precursor Insulin-like growth factor binding protein 6 precursor Insulin-like growth factor binding protein 7 precursor Insulin-like growth factor binding protein complex acid labile chain precursor Integral membrane protein 2B Inter-alpha-trypsin inhibitor heavy chain H1 precursor ISLR precursor JAW1-related protein MRVI1B short isoform Kallikrein 6 precursor Kappa 1 light chain variable region Keratin 1 Keratin 10 Keratin 10 Keratin 16 Keratin 1b Keratin 6 IRS4 Keratin 6C Keratin 6L Keratin 7 Keratin 9 Keratin, type I cytoskeletal 10 Keratin, type II cuticular HB1 Keratin, type II cuticular HB4 Keratin, type II cytoskeletal 2 epidermal Keratin, type II cytoskeletal 3 Keratin, type II cytoskeletal 4 Keratin, type II cytoskeletal 5


324


Protein Name

IPI Address

Keratin, type II cytoskeletal 6D Keratin, type II cytoskeletal 6F KIAA0170 protein KIAA0284 protein KIAA0319 protein KIAA0387 protein KIAA0523 protein KIAA0584 protein KIAA0644 protein KIAA0661 protein KIAA0792 protein KIAA1009 protein KIAA1061 protein KIAA1291 protein KIAA1318 protein KIAA1373 protein KIAA1417 protein KIAA1458 protein KIAA1503 protein KIAA1529 protein KIAA1838 protein KIAA1877 protein KIAA1922 protein KRT17 protein KRT8 protein Kunitz-type protease inhibitor 2 precursor Lactate dehydrogenase B Latent TGF-beta binding protein-4 Latent transforming growth factor beta binding protein 2 Latent transforming growth factor-beta binding protein 4 Latent transforming growth factor-beta-binding protein 2 precursor Leishmanolysin-like peptidase, variant 2 Leucine zipper protein 1 Leucine-rich alpha-2-glycoprotein precursor L-FILIP Limbic system-associated membrane protein precursor LIR-D1 LISCH protein isoform 1 LISCH protein, isoform 2 LOC123872 protein Lumican precursor Ly-6/neurotoxin-like protein 1 precursor Lymphocyte antigen Ly-6H precursor Lysosomal alpha-glucosidase precursor Lysozyme C precursor Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA MANSC domain containing protein 1 precursor Matrix Gla-protein precursor Metalloproteinase inhibitor 2 precursor



325

Protein Name

IPI Address

MHC class I antigen precursor MIC2L1 isoform E3’-E4’-E3-E4 Mimecan precursor MOG protein Monocyte differentiation antigen CD14 precursor Multiple coagulation factor deficiency protein 2 precursor Multiple EGF-like-domain protein 4 Multiple PDZ domain protein Myelin-associated glycoprotein precursor Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin light chain variable region Myristoylated alanine-rich protein kinase C substrate N-acetylgalactosamine-4-O-sulfotransferase N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase NACHT-, LRR- and PYD-containing protein 7 Natural killer cell-specific antigen KLIP1 Nebulin Nebulin Neural cell adhesion molecule Neural cell adhesion molecule 1, 140 kDa isoform precursor Neural cell adhesion molecule 2 Neural-cadherin precursor Neurexin 1-alpha precursor Neurexophilin 1 precursor Neurexophilin 4 Neuroblastoma suppressor of tumorigenicity 1 precursor Neuroblastoma-amplified protein Neurocan core protein precursor Neuroendocrine convertase 1 precursor Neurofascin isoform 2 Neurofilament triplet H protein Neuroligin 2 precursor Neuron navigator 1 Neuron specific protein family member 1 Neuronal pentraxin I precursor Neuronal pentraxin receptor Neuronal pentraxin receptor isoform 1 Neuronal potassium channel alpha subunit Neuropeptide Y precursor Neurosecretory protein VGF precursor Nidogen-2 precursor Nociceptin precursor Nogo receptor-like 3


326


Protein Name

IPI Address

Notch homolog 2 NOV protein homolog precursor Novel protein Novel protein Novel protein Novex-3 titin isoform Nucleobindin 1 precursor Obscurin P15 protein P15 protein P60 Paraoxonase 1 Parvalbumin PBP family protein precursor Peptidylglycine alpha-amidating monooxygenase isoform B preproprotein Pericentrin 2 Periplakin PF6 Phosphatidylcholine-sterol acyltransferase precursor Phosphatidylinositol 3-kinase-related protein kinase Pigment epithelium-derived factor precursor Plasma glutathione peroxidase precursor Plasma protease C1 inhibitor precursor Plasma retinol-binding protein precursor Plasma serine protease inhibitor precursor Plasminogen precursor Platelet-derived growth factor beta isoform 2, preproprotein Plectin 10 Plectin 2 Plectin 3 Plectin 6 Plectin 8 PLXDC2 protein Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1 Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 3 Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 PREDICTED: dynein, cytoplasmic, heavy polypeptide 2 PREDICTED: FLJ46675 protein PREDICTED: G2 protein PREDICTED: hemicentin-2 PREDICTED: hypothetical protein FLJ13305 PREDICTED: hypothetical protein XP 291007 PREDICTED: hypothetical protein XP 373647 PREDICTED: hypothetical protein XP 373957 PREDICTED: hypothetical protein XP 375869 PREDICTED: hypothetical protein XP 498788 PREDICTED: KIAA1076 protein PREDICTED: KIAA1836 protein PREDICTED: leucine rich repeat containing 4B



327

Protein Name

IPI Address

PREDICTED: lunatic fringe homolog PREDICTED: MAX dimerization protein 5 PREDICTED: similar to ataxin-1 ubiquitin-like interacting protein PREDICTED: similar to BA92K2.2 (similar to ubiquitin) PREDICTED: similar to Beta-1,3-N-acetylglucosaminyltransferase lunatic fringe (O-fucosy PREDICTED: similar to centromeric protein E (CENP-E protein) PREDICTED: similar to contains transmembrane (TM) region PREDICTED: similar to FKSG30 PREDICTED: similar to hypothetical protein PREDICTED: similar to keratin, type I cytoskeletal 18 (cytokeratin 18) (K18) (CK 18) PREDICTED: similar to KIAA1501 protein PREDICTED: similar to KIAA1501 protein PREDICTED: similar to KIAA1693 protein PREDICTED: similar to phosphatidylethanolamine-binding protein (PEBP) (prostatic bindin) PREDICTED: similar to POTE2A PREDICTED: similar to pregnancy specific beta-1-glycoprotein 7 PREDICTED: similar to ribosomal protein L7 PREDICTED: similar to ribosomal protein L7 PREDICTED: similar to ribosomal protein S27a PREDICTED: similar to RIKEN cDNA 4732495G21 gene PREDICTED: similar to RIKEN cDNA 4930583C14 PREDICTED: similar to tripartite motif-containing 43 PREDICTED: similar to ZGC:66168 protein Prepro-alpha2(I) collagen precursor Preprotachykinin B Prion protein Probable endonuclease KIAA0830 precursor Probable G protein-coupled receptor 37 precursor Procollagen C-proteinase enhancer protein precursor Proenkephalin A precursor Proline-rich acidic protein ProSAAS precursor Prostaglandin-H2 D-isomerase precursor Protease, serine, 3 Protein FAM3C precursor Protein KIAA0494 Protein kinase C-binding protein NELL2 precursor Protein phosphatase 3 Protein tyrosine phosphatase, non-receptor type substrate 1 precursor Protein tyrosine phosphatase, receptor type, D isoform 2 precursor Protein tyrosine phosphatase, receptor type, D isoform 3 precursor Protein tyrosine phosphatase, receptor type, N polypeptide 2 isoform 2 precursor Protein tyrosine phosphatase, receptor type, sigma isoform 3 precursor Prothrombin precursor PRRG1 protein PTPRN2 protein Pyruvate kinase 3 isoform 2 Quiescin Ran binding protein 2


328


Protein Name

IPI Address

Ran-binding protein 2 Ras GTPase-activating protein 2 Ras GTPase-activating-like protein IQGAP1 Receptor-type tyrosine-protein phosphatase gamma precursor Receptor-type tyrosine-protein phosphatase-like N precursor Reticulocalbin 2 precursor Reticulon 4, isoform D Retinoblastoma-associated factor 600 Retinol binding protein 4, plasma Retinol binding protein 4, plasma Rho-associated protein kinase 1 RTN3-A1 RUN and TBC1 domain containing 3 SARG904 Scotin Scrapie-responsive protein 1 precursor Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activate) Secretogranin I precursor Secretogranin II precursor Secretogranin III precursor Selenium binding protein 1 Selenium-binding protein 1 Selenoprotein M precursor Semaphorin 7A precursor Semaphorin sem2 Serine protease inhibitor kazal-type 5 precursor Serine/threonine protein phosphatase 2B catalytic subunit, alpha isoform Serine/threonine-protein kinase PLK2 Serotransferrin precursor SERPINC1 protein SERPIND1 protein Serum albumin precursor Serum amyloid A-4 protein precursor Seven transmembrane helix receptor SH3 and multiple ankyrin repeat domains 2 isoform 1 Similar to peptide N-glycanase homolog SIN3B long isoform Single chain FV Single-chain FV SNC73 protein Sodium channel beta-3 subunit precursor Somatostatin precursor Sortilin 1, preproprotein Sortilin-related receptor precursor SPARC precursor SPARC-like 1 SPARC-like protein 1 precursor Spectrin beta chain, brain 4 Splice isoform 1 of 85 kDa calcium-independent phospholipase A2



329

Protein Name

IPI Address

Splice isoform 1 of acyl-CoA-binding protein Splice isoform 1 of amyloid beta A4 protein precursor Splice isoform 1 of amyloid-like protein 2 precursor Splice isoform 1 of astrotactin 1 Splice isoform 1 of basigin precursor Splice isoform 1 of brevican core protein precursor Splice isoform 1 of calcyclin-binding protein Splice isoform 1 of cannabinoid receptor 1 Splice isoform 1 of cartilage acidic protein 1 precursor Splice isoform 1 of CD44 antigen precursor Splice isoform 1 of complement factor B precursor Splice isoform 1 of complement factor H precursor Splice isoform 1 of connective tissue growth factor precursor Splice isoform 1 of contactin 1 precursor Splice isoform 1 of EGF-containing fibulin-like extracellular matrix protein 1 precursor Splice isoform 1 of engulfment and cell motility protein 1 Splice isoform 1 of fibrinogen alpha/alpha-E chain precursor Splice isoform 1 of fibrinogen gamma chain precursor Splice isoform 1 of fibronectin precursor Splice isoform 1 of fibulin-1 precursor Splice isoform 1 of GDNF family receptor alpha 2 precursor Splice isoform 1 of HLA class I histocompatibility antigen, Cw-16 alpha chain precursor Splice isoform 1 of insulin-like growth factor II precursor Splice isoform 1 of inter-alpha-trypsin inhibitor heavy chain H4 precursor Splice isoform 1 of latrophilin 1 precursor Splice isoform 1 of lysosome-associated membrane glycoprotein 2 precursor Splice isoform 1 of macrophage colony stimulating factor-1 precursor Splice isoform 1 of matrix metalloproteinase-17 precursor Splice isoform 1 of neural cell adhesion molecule 1, 120 kDa isoform precursor Splice isoform 1 of neurexin 2-alpha precursor Splice isoform 1 of neuroendocrine protein 7B2 precursor Splice isoform 1 of pantothenate kinase 2, mitochondrial precursor Splice isoform 1 of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 Splice isoform 1 of phospholipid transfer protein precursor Splice isoform 1 of platelet-derived growth factor, A chain precursor Splice isoform 1 of plectin 1 Splice isoform 1 of proactivator polypeptide precursor Splice isoform 1 of receptor-type tyrosine-protein phosphatase N2 precursor Splice isoform 1 of receptor-type tyrosine-protein phosphatase zeta precursor Splice isoform 1 of reticulon 1 Splice isoform 1 of reticulon 4 Splice isoform 1 of serine/threonine-protein kinase haspin Splice isoform 1 of serologically defined colon cancer antigen 1 Splice isoform 1 of stromal cell-derived factor 1 precursor Splice isoform 1 of SWI/SNF-related, matrix associated, actin-dependent regulator of of chromatin subfamily a containing dead/H box 1 Splice isoform 1 of testican-3 precursor Splice isoform 2 of acyl-CoA-binding protein Splice isoform 2 of alpha-mannosidase IIx

IPI00010182 IPI00006608 IPI00031030 IPI00022367 IPI00218019 IPI00456623 IPI00395627 IPI00009396 IPI00451624 IPI00305064 IPI00019591 IPI00029739 IPI00020977 IPI00029751 IPI00029658 IPI00219532 IPI00021885 IPI00021891 IPI00022418 IPI00296534 IPI00011732 IPI00472711 IPI00001611 IPI00294193 IPI00183445 IPI00009030 IPI00015881 IPI00008533 IPI00411478 IPI00007921 IPI00008944 IPI00171176 IPI00295252 IPI00022733 IPI00021833 IPI00014898 IPI00012503 IPI00334666 IPI00291099 IPI00003971 IPI00021766 IPI00397836 IPI00301618 IPI00413781 IPI00220119 IPI00478890 IPI00218836 IPI00220303

330


Protein Name

IPI Address

Splice isoform 2 of amyloid-like protein 2 precursor Splice isoform 2 of apoptotic protease activating factor 1 Splice isoform 2 of collagen alpha 1(XVIII) chain precursor Splice isoform 2 of connective tissue growth factor precursor Splice isoform 2 of contactin 1 precursor Splice isoform 2 of ectonucleotide pyrophosphatase/phosphodiesterase 2 Splice isoform 2 of far upstream element binding protein 1 Splice isoform 2 of fibrinogen alpha/alpha-E chain precursor Splice isoform 2 of HIV-1 Rev binding protein-like protein Splice isoform 2 of HpaII tiny fragments locus 9C protein Splice isoform 2 of insulin-like growth factor II precursor Splice isoform 2 of kininogen precursor Splice isoform 2 of latrophilin 1 precursor Splice isoform 2 of neural cell adhesion molecule L1 precursor Splice isoform 2 of neuroendocrine protein 7B2 precursor Splice isoform 2 of neuroligin 3 precursor Splice isoform 2 of neuronal cell adhesion molecule precursor Splice isoform 2 of osteopontin precursor Splice isoform 2 of olatelet-derived growth factor, A chain precursor Splice isoform 2 of proactivator polypeptide precursor Splice isoform 2 of receptor-type tyrosine-protein phosphatase N2 precursor Splice isoform 2 of sex hormone-binding globulin precursor Splice isoform 2 of signal-regulatory protein beta-2 precursor Splice isoform 2 of SPARC related modular calcium-binding protein 1 precursor Splice isoform 3 of A-kinase anchor protein 9 Splice isoform 3 of amyloid beta A4 protein precursor Splice isoform 3 of amyloid-like protein 2 precursor Splice isoform 3 of bullous pemphigoid antigen 1, isoforms 6/9/10 Splice isoform 3 of EGF-containing fibulin-like extracellular matrix protein 1 precursor Splice isoform 3 of integrin alpha-7 precursor Splice isoform 3 of myelin-oligodendrocyte glycoprotein precursor Splice isoform 3 of neuronal cell adhesion molecule precursor Splice isoform 3 of neurotrimin precursor Splice isoform 3 of peptidyl-glycine alpha-amidating monooxygenase precursor Splice isoform 3 of proactivator polypeptide precursor Splice isoform 3 of receptor-type tyrosine-protein phosphatase S precursor Splice isoform 3 of SH3 and multiple ankyrin repeat domains protein 1 Splice isoform 3 of WAP four-disulfide core domain protein 2 precursor Splice isoform 4 Of EGF-containing fibulin-like extracellular matrix protein 1 precursor Splice isoform 4 of fibulin-1 precursor Splice isoform 4 of integrin beta-1 precursor Splice isoform 4 of nesprin 1 Splice isoform 4 of nuclear autoantigen Sp-100 Splice isoform 4 of osteopontin precursor Splice isoform 4 of seizure 6-like protein precursor Splice isoform 5 of amyloid beta A4 protein precursor Splice isoform 6 of amyloid beta A4 protein precursor Splice isoform 7 of amyloid beta A4 protein precursor Splice isoform 7 of myelin-oligodendrocyte glycoprotein precursor



331

Protein Name

IPI Address

Splice isoform 8 of amyloid beta A4 protein precursor Splice isoform 8 of myelin-oligodendrocyte glycoprotein precursor Splice isoform 9 of amyloid beta A4 protein precursor Spondin 1 precursor SPUF protein precursor Sulfatase 2 isoform B precursor Superoxide dismutase 1, soluble Synaptotagmin-11 TA p63 alpha TBC1 domain family member 10 T-cell activation Rho GTPase activating protein Tenascin-R Testican-1 precursor Testican-2 precursor Tetranectin precursor Tetratricopeptide repeat protein 14 Thioredoxin Threonine aspartase 1 Thy-1 membrane glycoprotein precursor Thymosin, beta 10 Thymosin, beta 4 Thymosin, beta 4, Y chromosome Thymosin-like 3 Thyroxine-binding globulin precursor Titin Titin isoform novex-1 Titin, heart isoform N2-B Toll-like receptor 7 precursor Transforming growth factor-beta induced protein IG-H3 precursor Transient receptor potential cation channel subfamily M member 7 Transthyretin precursor Tripartite motif protein 26 Type I inner root sheath specific keratin 25 irs3 Type XV collagen Ubiquitin 4 Ubiquitin and ribosomal protein L40 precursor Ubiquitin and ribosomal protein S27a precursor Usher syndrome 1C binding protein 1 Uveal autoantigen Vacuolar ATP synthase subunit S1 precursor VGFG2573 VH3 protein Villin 1 Vitamin D-binding protein precursor Vitamin K-dependent protein S precursor Vitronectin precursor VPS10 domain-containing receptor SORCS2 precursor VPS10 domain-containing receptor SORCS3 precursor WAP four-disulfide core domain protein 1 precursor


332


Protein Name

IPI Address

Werner helicase interacting protein WUGSC:DJ515N1.2 protein WUGSC:H DJ0747G18.3 protein WW domain containing adaptor with coiled-coil WW domain-containing adapter with a coiled-coil region, isoform 1 Xylosyltransferase I Zona pellucida sperm-binding protein 2 precursor


Appendix II (Proteins identified by single peptide)

Protein Name

IPI Address

101 kDa protein 101 kDa protein 11 kDa protein 110 kDa protein 130 kD Golgi-localized phosphoprotein 14 kDa protein 141 kDa protein 15 kDa protein 15 kDa protein 25 kDa protein 25 kDa protein 28S ribosomal protein S18c, mitochondrial precursor 344 kDa protein 376 kDa protein 39 kDa protein 39 kDa protein 47 kDa protein 48 kDa protein 65 kDa protein 66 kDa protein 67 kDa protein 71 kDa protein 73 kDa protein 82 kDa protein 84 kDa protein 90 kDa protein 97 kDa protein 99 kDa protein ABC transporter ABCA7 Acetoacetyl-CoA synthetase ACSL6 protein Actin, alpha cardiac Actin, aortic smooth muscle

IPI00478742 IPI00291316 IPI00382841 IPI00473056 IPI00004962 IPI00478089 IPI00478948 IPI00413031 IPI00413387 IPI00412608 IPI00477989 IPI00007049 IPI00479834 IPI00479143 IPI00070070 IPI00479497 IPI00479602 IPI00414747 IPI00414018 IPI00479085 IPI00332849 IPI00477128 IPI00412783 IPI00478238 IPI00412853 IPI00478692 IPI00472544 IPI00414260 IPI00293895 IPI00217272 IPI00384110 IPI00023006 IPI00008603


333

Protein Name

IPI Address

Activating receptor pilrbeta ADAM 10 precursor ADAMTS-1 precursor Adapter-related protein complex 1 gamma 1 subunit Adipsin/complement factor D precursor Adlican ADM precursor A-gamma globin Agrin precursor ALMS1 protein AlphA 1 type XIII collagen isoform 3 AlphA 3 type VI collagen isoform 4 precursor Alpha tachykinin 3 variant 2 Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase Alpha-1-acid glycoprotein 1 precursor Alpha-fetoprotein precursor Alpha-ketoglutarate dehydrogenase complex dihydrolipoyl succinyltransferase Alu subfamily SQ sequence contamination warning entry Aminomethyltransferase, mitochondrial precursor Angiopoietin-related protein 2 precursor Angiotensin I converting enzyme, isoform 3 Ankyrin repeat and SOCS box protein 2 Ankyrin repeat domain protein 9 Antigen MLAA-20 APG7L protein Apolipoprotein B-100 precursor Apolipoprotein F precursor ARHGAP8 protein Aspartate aminotransferase 1 ATP-binding cassette, sub-family A (ABC1), member 1 ATP-binding cassette, sub-family A, member 1 ATP-dependent RNA helicase DDX24 Atrial/embryonic alkali myosin light chain Autosomal highly conserved protein AXL receptor tyrosine kinase, isoform 1 BA231F10.1 Bactericidal/permeability-increasing protein-like 3 precursor Beta-1,4 N-acetylgalactosaminyltransferase Beta-hexosaminidase alpha chain precursor Beta-neoendorphin-dynorphin precursor BMP and activin membrane-bound inhibitor homolog precursor Bone morphogenetic protein 15 precursor Bone specific CMF608 Brain protein Brain-derived neurotrophic factor precursor Brain-specific angiogenesis inhibitor 1 precursor Brain-specific angiogenesis inhibitor 3 precursor Bromodomain protein CELTIX1 Bullous pemphigoid antigen 1 isoform 1


334


Protein Name

IPI Address

Bullous pemphigoid antigen 1 isoform 1EB precursor C17orf28 protein C1D protein C1orf40 protein C21orf258 protein C9orf86 protein Cadherin 11, type 2, isoform 1 preproprotein Cadherin-22 precursor Calcitonin gene-related peptide I precursor Calcium binding protein Cab45 precursor Calcium/calmodulin-dependent protein kinase II delta, isoform 1 Calmin Calmodulin 2 Calreticulin precursor Calsyntenin-3 precursor CANT1 protein Cappuccino protein homolog Cathepsin F precursor Cathepsin L precursor Cathepsin S precursor Catsper4 Cell recognition protein CASPR4 Cell surface glycoprotein MUC18 precursor Centrosome protein Cep63 Centrosome-associated protein 350 Cerebellin 3 precursor Chemokine (C-X-C motif) ligand 16 Chondroitin sulfate proteoglycan 5-III Chromosome 10 open reading frame 88 Chromosome 6 open reading frame 152 Chromosome 9 open reading frame 140 Chronic myelogenous leukemia tumor antigen 66 Coagulation factor X precursor Coagulation factor XII precursor Cohesin subunit SA-1 Coiled-coil domain containing protein 9 Collagen alpha 1 Collagen alpha 1(III) chain precursor Collagen alpha 1(XV) chain precursor Complement C1q tumor necrosis factor-related protein 4 precursor Component of oligomeric Golgi complex 6 CSRV314 Cystatin M precursor Cytochrome P450 1A1 Cytokine-like protein C17 precursor DEAH (Asp-Glu-Ala-His) box polypeptide 29 Delta globin Dermatopontin precursor Dermcidin precursor



335

Protein Name

IPI Address

Dermokine-beta Dipeptidyl peptidase-like protein 2 Dipeptidyl-peptidase II precursor DJ1003J2.3.1 DJ1042K10.2.2 DJ1119A7.3 DJ153G14.2 DJ68D18.1.2 DJ788L20.2 DJ977L11.1 DKFZp434L142 protein DKFZP564O243 protein DNA cytosine methyltransferase 3 alpha, isoform A DNA excision repair protein ERCC-6 DNA-directed RNA polymerase I largest subunit DNA-repair protein XRCC2 DRIM protein Dyskerin Endothelin 3, isoform 2 preproprotein Ephrin A1 isoform B precursor Ephrin receptor ephB3 Epsilon globin Erythrocyte membrane protein band 4.2 Estrogen receptor binding protein Exostosin-like 3 F1Fo-ATP synthase complex Fo membrane domain G subunit FAM31B protein Fanconi anemia group E protein Fas apoptotic inhibitory molecule 2 FCGR3A protein Fibromodulin FLJ00006 protein FLJ00172 protein FLJ00179 protein FLJ00199 protein FLJ00239 protein FLJ00332 protein FLJ11029 protein FLJ34512 protein Follistatin-related protein 1 precursor Formin 2 Formin-binding protein 17 Frizzled-related protein precursor Full-length cDNA 5-prime end of clone CS0DJ009YL13 of T cells Full-length cDNA clone CS0DM007YO13 of fetal liver of homo sapiens FXYD6 FYVE and coiled-coil domain containing 1 G protein-coupled sphingolipid receptor G4 protein


336


Protein Name

IPI Address

GAJ Gamma tachykinin 3 variant 2 Gamma-synuclein Gelsolin precursor Glutaminyl-peptide cyclotransferase precursor Glutaredoxin (thioltransferase) Glyceraldehyde-3-phosphate dehydrogenase Glycerol kinase, isoform A Golgi apparatus protein 1 Golgin-67 isoform A Grb10 interacting GYF protein 1 Grb10 interacting GYF protein 2 Gremlin Guanylin precursor HBB protein Heat shock 10kDa protein 1 (chaperonin 10) HEJ1 Hematopoietic PBX-interacting protein Hemoglobin beta Hepatocellular carcinoma associated protein TB6 Heterogeneous nuclear ribonucleoprotein L isoform B HEXIM1 protein Hexokinase, type II HGF activator like protein HGS RE408 HIV TAT specific factor 1 HRPE773 HSPC009 HSPC098 Hus1+-like protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein



337

Protein Name

IPI Address

Hypothetical protein Hypothetical protein Hypothetical protein DKFZp434A2017 Hypothetical protein DKFZp434C011 Hypothetical protein DKFZP434J0113 Hypothetical protein DKFZp434K1421 Hypothetical protein DKFZp434P097 Hypothetical protein DKFZp434P1219 Hypothetical protein DKFZp547D2210 Hypothetical protein DKFZp547N1615 Hypothetical protein DKFZp586K2123 Hypothetical protein DKFZp666G229 Hypothetical protein DKFZp686A06175 Hypothetical protein DKFZp686C086 Hypothetical protein DKFZp686C195 Hypothetical protein DKFZp686D0623 Hypothetical protein DKFZp686D0880 Hypothetical protein DKFZp686G09165 Hypothetical protein DKFZp686H14204 Hypothetical protein DKFZp686H22230 Hypothetical protein DKFZp686L13193 Hypothetical protein DKFZp686N18114 Hypothetical protein DKFZp686O0186 Hypothetical protein DKFZp761D171 Hypothetical protein DKFZp761F0118 Hypothetical protein DKFZp761G128 Hypothetical protein DKFZp761M0817 Hypothetical protein DKFZp781A0122 Hypothetical protein FLJ10650 Hypothetical protein FLJ10871 Hypothetical protein FLJ10955 Hypothetical protein FLJ12133 Hypothetical protein FLJ12666 Hypothetical protein FLJ13110 Hypothetical protein FLJ13409 Hypothetical protein FLJ13459 Hypothetical protein FLJ13782 Hypothetical protein FLJ13813 Hypothetical protein FLJ14456 Hypothetical protein FLJ14494 Hypothetical protein FLJ14714 Hypothetical protein FLJ16032 Hypothetical protein FLJ16127 Hypothetical protein FLJ16417 Hypothetical protein FLJ20055 Hypothetical protein FLJ21011 Hypothetical protein FLJ21156 Hypothetical protein FLJ21415 Hypothetical protein FLJ21816


338


Protein Name

IPI Address

Hypothetical protein FLJ22474 Hypothetical protein FLJ23420 Hypothetical protein FLJ25224 Hypothetical protein FLJ25690 Hypothetical protein FLJ30356 Hypothetical protein FLJ31401 Hypothetical protein FLJ32363 Hypothetical protein FLJ32451 Hypothetical protein FLJ32800 Hypothetical protein FLJ32842 Hypothetical protein FLJ34512 Hypothetical protein FLJ34922 Hypothetical protein FLJ38419 Hypothetical protein FLJ38522 Hypothetical protein FLJ39374 Hypothetical protein FLJ39963 Hypothetical protein FLJ40941 Hypothetical protein FLJ41598 Hypothetical protein FLJ42730 Hypothetical protein FLJ43795 Hypothetical protein FLJ44006 Hypothetical protein FLJ44069 Hypothetical protein FLJ44161 Hypothetical protein FLJ44241 Hypothetical protein FLJ44324 Hypothetical protein FLJ45140 Hypothetical protein FLJ45264 Hypothetical protein FLJ45525 Hypothetical protein FLJ45715 Hypothetical protein FLJ45736 Hypothetical protein FLJ46550 Hypothetical protein FLJ46675 Hypothetical protein FLJ46747 Hypothetical protein FLJ90005 Hypothetical protein FLJ90091 Hypothetical protein FLJ90551 Hypothetical protein FLJ90661 Hypothetical protein LOC113174 Hypothetical protein LOC122618 Hypothetical protein LOC90624 Hypothetical protein MGC26885 Hypothetical protein MGC29784 Hypothetical protein PIK3CG Hypothetical protein PSEC0200 Hypothetical protein PSEC0250 ICBP90 binding protein 1 Ig heavy chain V-II region SESS precursor Ig heavy chain V-III region BUT Ig heavy chain V-III region GA



339

Protein Name

IPI Address

Ig heavy chain V-III region WAS Ig kappa chain V-I region BAN Ig kappa chain V-I region HK102 precursor Ig kappa chain V-III region VG precursor Ig kappa chain V-III region VH precursor Ig kappa chain V-IV region B17 precursor Ig lambda chain V-III region SH Ig lambda chain V-IV region Hil IL-17RC Immunoglobulin-like domain protein MGC33530 precursor Import inner membrane translocase subunit TIM44, mitochondrial precursor Importin 9 Inhibin beta A chain precursor Inositol polyphosphate-5-phosphatase F Insulin receptor tyrosine kinase substrate Insulin-like growth factor IB precursor Insulinoma-glucagonoma protein 20 splice variant 2 Integral membrane protein 2B Inter-alpha trypsin Inhibitor heavy chain precursor 5 Isoform 2 Inter-alpha-trypsin inhibitor heavy chain H2 precursor Intercellular adhesion molecule-5 precursor Interleukin 17 receptor C, isoform 3 Interleukin-1 receptor-associated kinase-like 2 IQ motif containing E Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor Isocitrate dehydrogenase [NADP] cytoplasmic Kainate receptor subunit KA2a KARCA1 protein Kelch/ankyrin repeat containing cyclin A1 interacting protein Keratin b20 KHSRP protein KIAA0300 protein KIAA0323 protein KIAA0351 protein KIAA0372 protein KIAA0443 protein KIAA0477 protein KIAA0523 protein KIAA0663 protein KIAA1185 protein KIAA1204 protein KIAA1265 protein KIAA1274 protein KIAA1384 protein KIAA1450 protein KIAA1604 protein KIAA1640 protein KIAA1730 protein KIAA1840 protein


340


Protein Name

IPI Address

KIAA1946 KSS splice variant b Laminin alpha-1 chain precursor Laminin gamma-1 chain precursor LAR Latent transforming growth factor beta binding protein 1 isoform LTBP-1L Latent transforming growth factor beta binding protein, isoform 1L precursor Leukemia-associated protein with a CXXC domain Leukocyte receptor cluster (LRC) member 1 Line-1 repeat mRNA with 2 open reading frames Lipopolysaccharide-binding protein precursor Liprin-alpha 2 LOC374654 protein LP2209 L-plastin LTLL9335 Lysosomal-associated multitransmembrane protein Macrophage colony stimulating factor I receptor precursor Major prion protein precursor Mammalian ependymin related protein 1 Mannosidase, alpha, class 1B, member 1 Mannosyl-oligosaccharide 1,2-alpha-mannosidase IC MAP-kinase activating death domain-containing protein isoform a Matrin 3 Mckusick-Kaufman/Bardet-Biedl syndromes putative chaperonin MDM1 protein Megakaryocyte-associated tyrosine-protein kinase Melanoma derived growth regulatory protein precursor Metabotropic glutamate receptor 3 precursor Metalloproteinase inhibitor 1 precursor Metallothionein-III MGAT3 protein MIC2L1 isoform E3-E4 Microfibrillar-associated protein 5 precursor Microsomal signal peptidase 18 kDa subunit Microtubule-associated protein 1B isoform 2 Middle-chain acyl-CoA synthetase1 Minichromosome maintenance protein 10 isoform 1 Mitochondrial ribosomal protein L48 Mitogen-activated protein kinase 12 Mitotic kinesin-related protein Molybdenum cofactor synthesis protein 2 small subunit Monocarboxylate transporter 3 MOP-4 Mosaic serine protease Mothers against decapentaplegic homolog 4 MSFL2541 Mu-crystallin homolog Muellerian inhibiting factor precursor



341

Protein Name

IPI Address

Multi-functional protein MFP Multiple inositol polyphosphate phosphatase Muscle-cadherin precursor Muscle-type acylphosphatase 2 Myelin associated glycoprotein isoform B precursor Myelin P0 protein precursor Myelin protein zero Myosin IF Myosin-reactive immunoglobulin heavy chain variable region Myosin-reactive immunoglobulin kappa chain variable region N-acetyltransferase 5 isoform B NDST2 protein Nebulin-related anchoring protein Nectin-like protein 3 NEFL protein Neural cell adhesion molecule 1 Neural proliferation differentiation and control protein-1 precursor Neurexin 1-beta precursor Neurexophilin 4 precursor Neuritin Neuroendocrine convertase 2 precursor Neurogenic locus notch homolog protein 2 precursor Neurogenin 3 Neurotrypsin precursor NICE-4 protein NIPA1 protein Nyctalopin precursor Olfactory receptor 51Q1 Optic atrophy 1 isoform 4 Optineurin isoform 1 Ornithine decarboxylase antizyme 2 Orthopedia Osteomodulin precursor OTTHUMP00000021593 OTTHUMP00000021980 OTTHUMP00000031659 OTTHUMP00000042410 Oxidored-nitro domain-containing protein Oxytocin-neurophysin 1 precursor Paired-like homeobox protein PEPP-1 PCPB protein Peptidyl-prolyl cis-trans isomerase C Peroxiredoxin 2 Phosphatidylinositol transfer protein, cytoplasmic 1, isoform B PHYHD1 protein PKY protein kinase PLC-zeta Pleiotrophin precursor PNAS-138


342


Protein Name

IPI Address

Podocalyxin-like protein Polymeric-immunoglobulin receptor precursor Potassium voltage-gated channel subfamily C member 1 Potassium voltage-gated channel subfamily KQT member 3 Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 PPIB protein PR domain containing 10 isoform 1 PREDICTED: C219-reactive peptide PREDICTED: chromosome 14 open reading frame 125 PREDICTED: chromosome 20 open reading frame 82 PREDICTED: hypothetical protein LOC150368 PREDICTED: hypothetical protein XP 211408 PREDICTED: hypothetical protein XP 373555 PREDICTED: hypothetical protein XP 373915 PREDICTED: hypothetical protein XP 373979 PREDICTED: hypothetical protein XP 374010 PREDICTED: hypothetical protein XP 374046 PREDICTED: hypothetical protein XP 374095 PREDICTED: hypothetical protein XP 374333 PREDICTED: hypothetical protein XP 378700 PREDICTED: hypothetical protein XP 379029 PREDICTED: hypothetical protein XP 379306 PREDICTED: hypothetical protein XP 498568 PREDICTED: hypothetical protein XP 499091 PREDICTED: hypothetical protein XP 499305 PREDICTED: KIAA0367 protein PREDICTED: KIAA0527 protein PREDICTED: KIAA0819 protein PREDICTED: KIAA1337 protein PREDICTED: KIAA1543 PREDICTED: KIAA1856 protein PREDICTED: odz, odd Oz/ten-m homolog 2 PREDICTED: odz, odd Oz/ten-m homolog 3 PREDICTED: similar to 28 kDa heat- and acid-stable phosphoprotein (PDGF-associated protein) PREDICTED: similar to 40S ribosomal protein S16 PREDICTED: similar to 60S ribosomal protein L23a PREDICTED: similar to adrenoleukodystrophy protein (ALDP) PREDICTED: similar to anaphase promoting complex subunit 1 PREDICTED: similar to asparagine synthetase PREDICTED: similar to CCR4-NOT transcription complex, subunit 6-like PREDICTED: similar to CG3047-PA PREDICTED: similar to chloride intracellular channel protein 4 (intracellular chloride) PREDICTED: similar to DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 PREDICTED: similar to fatty acid-binding protein, epidermal (E-FABP) (psoriasis-associated fatty acid-binding protein homolog) PREDICTED: similar to germ and embryonic stem cell enriched protein STELLA PREDICTED: similar to glutathione S-transferase Mu 5 (GSTM5-5) (GST class-Mu 5) PREDICTED: similar to golgin-67 isoform C PREDICTED: similar to heat shock 10kDa protein 1 (chaperonin 10)



343

Protein Name

IPI Address

PREDICTED: similar to histidine-rich glycoprotein precursor (histidine-proline rich glycoprotein) PREDICTED: similar to hypothetical protein A830023L05 PREDICTED: similar to hypothetical protein BC005730 PREDICTED: similar to matrilin 2 precursor PREDICTED: similar to melanoma antigen, family A, 10 PREDICTED: similar to pre-mRNA splicing SR protein related (68.2 kD) (rsr-1) PREDICTED: similar to ribosome biogenesis protein BMS1 homolog PREDICTED: similar to RIKEN cDNA 4930539E08 PREDICTED: similar to SURF6 protein PREDICTED: similar to tumor necrosis factor, alpha-induced protein 2 PREDICTED: similar to ZNF43 protein PREDICTED: similar to zonadhesin PREDICTED: zinc finger protein 469 Pregnancy-specific beta-1-glycoprotein 8 precursor PRO1787 Profilin 2 isoform A Profilin-3 Progesterone receptor membrane component 1 Progesterone-induced blocking factor 1 Prolargin precursor Proline-rich protein 4 precursor Prolylcarboxypeptidase isoform 2 Prostate tumor overexpressed gene 1 Prostatic binding protein Protease inhibitor H Protein C20orf98 Protein FAM38A Protein PRO1854 Protein tyrosine phosphatase domain containing 1 protein isoform 1 Protein tyrosine phosphatase, non-receptor type 14 Protein-L-isoaspartate (D-aspartate) O-methyltransferase Protein-tyrosine sulfotransferase 1 Protocadherin 1 isoform 2 precursor Protocadherin fat 2 precursor P-selectin glycoprotein ligand 1 precursor PSMC3 protein PSST739 PTPL1-associated RhoGAP Purkinje cell protein 4 Putative 4 repeat voltage-gated ion channel Putative acyl-CoA thioester hydrolase CGI-16 Putative alpha-mannosidase C1orf22 Putative secreted ligand Putative secretory protein Pyruvate kinase 3 isoform 1 QVSK201 RAS p21 protein activator 3 Ras-related protein Rap-2B


344


Protein Name

IPI Address

Receptor-interacting serine/threonine-protein kinase 2 Replication protein A 70 kDa DNA-binding subunit Retbindin RGD, leucine-rich repeat, tropomodulin and proline-rich containing protein Rho guanine nucleotide exchange factor 1 isoform 1 Rho/rac-interacting citron kinase Rho/rac guanine nucleotide exchange factor (GEF) 2 Rho-GTPase activating protein 10 Rho-GTPase-activating protein 5 Ribonuclease 4 precursor Ribosomal protein L39 Ribosomal protein L3-like Ribosomal protein L7 Ribosome biogenesis protein BMS1 homolog RNA-binding protein 5 Rotatin SAA1 protein Salvador homolog 1 protein Sarco/endoplasmic reticulum Ca 2+ -ATPase isoform D SAYY8238 SCMH1 protein Selenoprotein P precursor SEMA3B protein Serine protease HTRA1 precursor Serine protease inhibitor, Kazal type, 5 Serine/threonine-protein kinase H1 Seven transmembrane helix receptor SH3 domain-binding glutamic acid-rich-like protein SH3-domain GRB2-like 1 Sia-alpha-2,3-Gal-beta-1,4-GlcNAc-R:alpha 2,8-sialyltransferase Similar to ecotropic viral integration site 5; neuroblastoma stage 4S gene Similar to expressed sequence AI593442 Similar to phospholipase C, beta 3 Similar to portion of neuronal pentraxin i NPX1 or NP1 Similar to protein kinase C substrate SLAP SLC5A12 protein Small intestine SPAK-like kinase SNC66 protein Sodium/potassium-transporting ATPase alpha-2 chain precursor Sortilin precursor Spir-2 protein Splice isoform 1 of activating signal cointegrator 1 complex subunit 3 Splice isoform 1 of ADAMTS-16 precursor Splice isoform 1 of ADAMTS-2 precursor Splice isoform 1 of adapter-related protein complex 3 beta 1 subunit Splice isoform 1 of adapter-related protein complex 3 delta 1 subunit Splice isoform 1 of aquaporin 4 Splice Isoform 1 of bone morphogenetic protein 1 precursor



345

Protein Name

IPI Address

Splice isoform 1 of calcium/calmodulin-dependent protein kinase type II alpha chain Splice isoform 1 of ccg1-interacting factor B Splice isoform 1 of collagen alpha 2(VI) chain precursor Splice isoform 1 of COP9 signalosome complex subunit 1 Splice isoform 1 of cullin homolog 4B Splice isoform 1 of cyclic-AMP-dependent transcription factor ATF-6 beta Splice isoform 1 of desmoplakin Splice isoform 1 of double-stranded RNA-specific adenosine deaminase Splice isoform 1 of dynein intermediate chain 1, cytosolic Splice isoform 1 of ecto-ADP-ribosyltransferase 3 precursor Splice isoform 1 of endothelin-3 precursor Splice isoform 1 of ERC protein 1 Splice isoform 1 of gamma-tubulin complex component 6 Splice isoform 1 of glutaryl-CoA dehydrogenase, mitochondrial precursor Splice isoform 1 of HpaII tiny fragments locus 9c protein Splice isoform 1 of inositol 1,4,5-trisphosphate receptor type 2 Splice isoform 1 of IQ calmodulin-binding motif containing protein 1 Splice isoform 1 of lysosomal trafficking regulator Splice isoform 1 of neuroligin 1 precursor Splice isoform 1 of neuropilin-1 precursor Splice isoform 1 of osteopontin precursor Splice isoform 1 of p130Cas-associated protein Splice isoform 1 of partitioning defective-6 homolog alpha Splice isoform 1 of pleckstrin homology domain containing family C member 1 Splice isoform 1 of polycystic kidney and hepatic disease 1 precursor Splice isoform 1 of protachykinin 1 precursor Splice isoform 1 of protein C21orf70 Splice Isoform 1 of putative polypeptide N-acetylgalactosaminyltransferase-like protein Splice isoform 1 of receptor-type tyrosine-protein phosphatase delta precursor Splice isoform 1 of rotavirus ’X’ associated non-structural protein Splice isoform 1 of serine/threonine-protein kinase RIPK4 Splice isoform 1 of SET binding factor 1 Splice isoform 1 of short transient receptor potential channel 6 Splice Isoform 1 of sodium/potassium-transporting ATPase alpha-1 chain precursor Splice isoform 1 of spectrin beta chain, brain 3 Splice isoform 1 of telomerase-binding protein EST1A Splice isoform 1 of tetratricopeptide repeat protein 7A Splice isoform 1 of transcription factor E2-alpha Splice isoform 1 of trans-Golgi network integral membrane protein 2 precursor Splice isoform 1 of tubby-like protein 4 Splice isoform 1 of ubiquitin carboxyl-terminal hydrolase 33 Splice isoform 1 of ubiquitin-conjugating enzyme E2 variant 1 Splice isoform 1 of UPF0338 protein NG5 Splice isoform 1 of vacuolar protein sorting 18 Splice isoform 1 of voltage-dependent N-type calcium channel alpha-1B subunit Splice isoform 1 of zinc finger DHHC domain containing protein 13 Splice isoform 10 of integrin alpha-7 precursor Splice isoform 11 of integrin alpha-7 precursor Splice isoform 2 of ADAMTS-16 precursor


346


Protein Name

IPI Address

Splice isoform 2 of ADAMTS-2 precursor Splice isoform 2 of amphiphysin Splice isoform 2 of angiogenic factor VG5Q Splice isoform 2 of apolipoprotein L1 precursor Splice isoform 2 of arfaptin 1 Splice isoform 2 of basigin precursor Splice isoform 2 of bone morphogenetic protein 1 precursor Splice isoform 2 of bromodomain adjacent to zinc finger domain protein 1A Splice isoform 2 of cadherin-11 precursor Splice isoform 2 of calcium/calmodulin-dependent protein kinase type II alpha chain Splice isoform 2 of canalicular multispecific organic anion transporter 2 Splice isoform 2 of CCR4-NOT transcription complex subunit 4 Splice isoform 2 of collagen alpha 2(VI) chain precursor Splice isoform 2 of collagen alpha 3(VI) chain precursor Splice isoform 2 of complement factor H precursor Splice isoform 2 of cullin homolog 1 Splice isoform 2 of development and differentiation-enhancing factor 2 Splice isoform 2 of EGF-containing fibulin-like extracellular matrix protein 1 precurso Splice isoform 2 of endothelin-3 precursor Splice isoform 2 of ephrin type-A receptor 5 precursor Splice isoform 2 of glutaryl-CoA dehydrogenase, mitochondrial precursor Splice isoform 2 of HLA class I histocompatibility antigen, Cw-16 alpha chain precursor Splice isoform 2 of ICOS ligand precursor Splice isoform 2 of insulin receptor precursor Splice isoform 2 of integrin alpha-7 precursor Splice isoform 2 of interleukin-12 receptor beta-2 chain precursor Splice isoform 2 of interleukin-18 binding protein precursor Splice isoform 2 of MAGUK p55 subfamily member 2 Splice isoform 2 of MAM domain-containing glycosylphosphatidylinositol anchor protein 1 Splice Isoform 2 of metabotropic glutamate receptor 8 precursor Splice isoform 2 of mitochondrial dicarboxylate carrier Splice isoform 2 of Myosin Va Splice isoform 2 of Myosin VIIa Splice isoform 2 of N-acetylmuramoyl-L-alanine amidase precursor Splice isoform 2 of neural cell adhesion molecule 1, 120 kDa isoform precursor Splice isoform 2 of NTF2-related export protein 2 Splice isoform 2 of oral-facial-digital syndrome 1 protein Splice isoform 2 of pleckstrin homology domain containing family C member 1 Splice isoform 2 of protachykinin 1 precursor Splice isoform 2 of protein-L-isoaspartate Splice isoform 2 of putative polypeptide N-acetylgalactosaminyltransferase-like protein Splice isoform 2 of retinoic acid receptor responder protein 1 Splice isoform 2 of roundabout homolog 2 precursor Splice isoform 2 of secretory carrier-associated membrane protein 1 Splice isoform 2 of sentrin-specific protease 6 Splice isoform 2 of sodium/potassium/calcium exchanger 2 precursor Splice isoform 2 of solute carrier family 26 member 6 Splice isoform 2 of stromal cell-derived factor 1 precursor Splice isoform 2 of T-cell surface glycoprotein E2 precursor



347

Protein Name

IPI Address

Splice isoform 2 of trans-Golgi network integral membrane protein 2 precursor Splice isoform 2 of ubiquilin 1 Splice isoform 2 of UDP-N-acetylglucosamine–peptide N-acetylglucosaminyltransferase 11 Splice isoform 2 of voltage-dependent N-type calcium channel alpha-1B subunit Splice isoform 3 of ADAMTS-9 precursor Splice isoform 3 of adapter-related protein complex 3 delta 1 subunit Splice isoform 3 of apoptotic protease activating factor 1 Splice isoform 3 of bone morphogenetic protein 1 precursor Splice isoform 3 of calcium/calmodulin-dependent protein kinase type II beta chain Splice isoform 3 of collagen alpha 2(VI) chain precursor Splice isoform 3 of dachshund homolog 2 Splice isoform 3 of latrophilin 3 precursor Splice isoform 3 of myosin XVIIIA Splice isoform 3 of osteopontin precursor Splice isoform 3 of receptor-type tyrosine-protein phosphatase delta precursor Splice isoform 3 of reelin precursor Splice isoform 3 of seizure 6-like protein precursor Splice isoform 3 of signal-regulatory protein beta-2 precursor Splice isoform 3 of solute carrier family 12 member 2 Splice isoform 3 of triggering receptor expressed on myeloid cells 2 precursor Splice isoform 3 of tuftelin Splice Isoform 3 of ubiquitin carboxyl-terminal hydrolase 6 Splice isoform 3 of versican core protein precursor Splice isoform 4 of integrin alpha-7 precursor Splice isoform 4 of peptidyl-glycine alpha-amidating monooxygenase precursor Splice isoform 4 of receptor-type tyrosine-protein phosphatase S precursor Splice isoform 5 of chordin precursor Splice isoform 5 of neuronal cell adhesion molecule precursor Splice isoform 5 of receptor-type tyrosine-protein phosphatase S precursor Splice isoform 6 of fibronectin precursor Splice isoform 6 of myelin-oligodendrocyte glycoprotein precursor Splice isoform 7 of calcium/calmodulin-dependent protein kinase type II beta chain SRPK2 protein ST6GalII protein Stem cell growth factor precursor Stromelysin-3 precursor Superoxide dismutase [Mn], mitochondrial precursor SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a1 isoform b Synaptotagmin VII Synaptotagmin-1 Synaptotagmin-4 Synphilin 1 T1 protein TAGLN protein TAR RNA loop binding protein TCN2 protein Testis-specific BRDT protein THAP domain protein 2 Tpr


348


Protein Name

IPI Address

Transcription elongation regulator 1 Transcription factor SL1 Transcription factor SOX-7 Transcription initiation factor IIE, alpha subunit Transcriptional activator SRCAP Transmembrane 7 superfamily member 1 Transmembrane protein 16B TRAP/Mediator complex component TRAP25 TRIF-related adapter molecule Triosephosphate isomerase 1 Tripeptidyl-peptidase I precursor tRNA-splicing endonuclease subunit SEN15 Tropomyosin 3 Trypsinogen C TSLC1-like 2 Tumor necrosis factor receptor superfamily member 19L precursor Tyrosine phosphatase zeta polypeptide 2 HTPZP2 Tyrosine-protein kinase CSK Ubiquitin carboxyl-terminal hydrolase 24 Ubiquitin carboxyl-terminal hydrolase isozyme L1 Ubiquitin-like protein fubi and ribosomal protein S30 precursor UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase 1, membrane-bound form Uncharacterized hematopoietic stem/progenitor cells protein MDS031 Unnamed secretory protein UTP14, U3 small nucleolar ribonucleoprotein, homolog A Utrophin Vesicular integral-membrane protein VIP36 precursor Vitamin K epoxide reductase complex, subunit 1-like 1 Voltage-dependent calcium channel gamma-6 subunit WD repeat membrane protein Werner helicase interacting protein, isoform 2 XA protein Zinc finger FYVE domain containing protein 28 Zinc finger protein Zinc finger protein 577 Zinc finger protein 95 homolog ZNF627 protein

IPI00247871 IPI00385907 IPI00027779 IPI00019977 IPI00009101 IPI00019017 IPI00033553 IPI00063213 IPI00329281 IPI00465028 IPI00298237 IPI00450071 IPI00479615 IPI00169276 IPI00176427 IPI00064377 IPI00472466 IPI00013212 IPI00398505 IPI00018352 IPI00019770 IPI00215767 IPI00020512 IPI00216914 IPI00107113 IPI00009329 IPI00009950 IPI00166079 IPI00011072 IPI00396243 IPI00102997 IPI00383520 IPI00288918 IPI00399361 IPI00013397 IPI00032316 IPI00029023

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