Diabetic nephropathy (DN) is a long-term complication of diabetes mellitus that leads to end-stage renal disease. Microalbuminuria is used for the early detection of diabetic renal damage, but such levels do not reflect the state of incipient DN precisely in type 2 diabetic patients because microalbuminuria develops in other diseases, necessitating more accurate biomarkers that detect incipient DN. Isobaric tags for relative and absolute quantification (iTRAQ) were used to identify urinary proteins that were differentially excreted in normoalbuminuric and microalbuminuric patients with type 2 diabetes where 710 and 196 proteins were identified and quantified, respectively. Some candidates were confirmed by 2-DE analysis, or validated by Western blot and multiple reaction monitoring (MRM). Specifically, some differentially expressed proteins were verified by MRM in urine from normoalbuminuric and microalbuminuric patients with type 2 diabetes, wherein alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen had excellent AUC values (0.849, 0.873, and 0.825, resp.). Moreover, we performed a multiplex assay using these biomarker candidates, resulting in a merged AUC value of 0.921. Although the differentially expressed proteins in this iTRAQ study require further validation in larger and categorized sample groups, they constitute baseline data on preliminary biomarker candidates that can be used to discover novel biomarkers for incipient DN.
Diabetes mellitus is a chronic disease with potentially devastating complications. For example, diabetes mellitus is associated with macrovascular complications, such as cardiovascular and cerebrovascular diseases, and microvascular complications, including diabetic nephropathy (DN) and retinopathy [
DN occurs in 15% to 25% of type 1 diabetic patients and 30% to 40% of type 2 diabetic patients [
The use of microalbuminuria to predict incipient DN, particularly in type 2 diabetic patients, is limited for several reasons [
Recently, to compare DN patients with non-DN patients, proteomic technologies have been developed to identify urinary marker candidates that are associated with the development of DN. Various proteomic approaches have been used for this purpose, including 2-DE, 2-DE DIGE, and SELDI-TOF [
To scan a comprehensive differential proteome for preliminary DN candidate biomarkers, we used a 4-plex isobaric tag for relative and absolute quantification (iTRAQ, 4-plex), allowing us to identify and quantify proteins in up to 4 samples [
Therefore, in this study, we used iTRAQ to identify and quantify differentially excreted urinary proteins in microalbuminuric versus normoalbuminuric type 2 diabetic patients and investigate the associations that would reflect the progress of DN. Afterward, those differentially excreted urinary proteins have been confirmed by 2-DE, followed by MALDI-TOF/TOF, or validated by Western blot and MRM.
Type 2 diabetic subjects (age ≥ 40 years) with or without microalbuminuria who were patients at the Diabetes Center of Seoul National University Hospital, Seoul, Republic of Korea, were enrolled in 2006. Microalbuminuria patients were randomly selected out of these outpatients, whereas normoalbuminuric patients were selected to be matched to age, sex, body mass index (BMI), and DM duration with microalbuminuric patients.
Forty-three subjects with diabetic retinopathy and persistent microalbuminuria formed the microalbuminuria group (MA). Persistent microalbuminuria was defined as an albumin : creatinine ratio (ACR) between 30 and 300 mg/g in 2 urine samples that were taken over 3 months. The normoalbuminuria group (NA) comprised subjects who had no diabetic retinopathy, did not use angiotensin inhibitors or angiotensin receptor blockers that lowered albuminuria, and showed no microalbuminuria in their urine in the past year (urinary albumin < 30 mg/g creatinine). Forty-three subjects formed the NA group.
There were no significant differences in age, sex, body mass index, or diabetes mellitus duration between the 2 study groups. Subjects with hematuria, uncontrolled hypertension (blood pressure ≥ 140/90 mm Hg), uncontrolled hyperglycemia (glycated hemoglobin A1c ≥ 8.5%), urinary tract infection, acute febrile illness, congestive heart failure, or malignancy were excluded. Individuals who were receiving peroxisome proliferator-activated receptor gamma agonists were also excluded. Midstream urine of spot urine samples were collected in sterile 50-mL tubes that contained 50
Urine albumin and creatinine were measured in spot urine samples by immunoturbidimetric method using the TIA Micro Alb Kit (Nittobo, Tokyo, Japan) and enzymatic creatinine assay (Roche, Mannheim, Germany), respectively, on a Hitachi 7170 autoanalyzer (Hitachi, Tokyo, Japan).
For the iTRAQ and 2-DE experiments, pooled urine samples, based on average albumin-to-creatinine ratios, were used; the clinical characteristics of the study subjects are summarized in Table
Clinical characteristics of normoalbuminuric (NA) and microalbuminuric (MA) type 2 diabetic patients.
Characteristics | NA 1a | MA 1a | NA 2b | MA 2b | NA 3c | MA 3c | NA 4d | MA 4d |
---|---|---|---|---|---|---|---|---|
( | ( | ( | ( | ( | ( | ( | ( | |
Gender (M/F) | 4/5 | 5/4 | 5/4 | 4/5 | 5/4 | 4/5 | 9/7 | 9/7 |
Mean age (years) | ||||||||
Duration of diabetes (years) | ||||||||
BMI (kg/m2) | ||||||||
Fasting plasma glucose (mg/dL) | ||||||||
HbA1C (%) | ||||||||
Blood urea nitrogen (mg/dL) | ||||||||
Serum creatinine (mg/dL) | ||||||||
Serum total cholesterol (mg/dL) | ||||||||
Serum HDL cholesterol (mg/dL) | ||||||||
Serum LDL cholesterol (mg/dL) | ||||||||
Serum triglycerides (mg/dL) | 177 ± 134.7 | |||||||
Albumin : creatinine ratio (mg/g) | 120.5 ± 70.7e | 82.6 ± 41.9f | ||||||
Pooled urine concentration (mg/mL) |
a–cSample sets for the three iTRAQ experiments, dsample sets for 2-DE, e
To prepare the protein samples, approximately 50 mL aliquots of normoalbuminuric and microalbuminuric urine were centrifuged at 3000 g for 30 min at 4°C. Supernatants were filtered through a 0.22
Aliquots of 100
In this study, 3 iTRAQ experiments were performed. The detailed iTRAQ labeling strategy is summarized for the specified NA/MA urine samples in Figure
Workflow of iTRAQ, 2-DE, Western blot, and MRM of the urinary proteome. For analysis of the urinary proteome, 3 iTRAQ experiments were performed, 2-DE, Western blot, and MRM were conducted to confirm and validate the iTRAQ results. iTRAQ experiments 1, 2, and 3 were performed, labeled (a) and (b), (c) and (d), and (e), respectively, wherein 3 biological replicates (labeled (a), (d), and (e), resp.), technical replicate 1 (labeled (a) and (c)), and technical replicate 2 (labeled (b) and (d)) were performed in microalbuminuric versus normoalbuminuric urine. 2-DE, Western blot, and MRM analysis of the urinary proteome were conducted using labeled (f), (g), and (h), respectively.
iTRAQ-labeled samples were subjected to LC-MS/MS at the National Instrumentation Center for Environmental Management, Seoul National University, and fractionated using strong cation exchange (SCX) chromatography, as follows. Dried samples were reconstituted in 500
Fractions were reconstituted in solvent A and injected into an LC-ESI-MS/MS system. LC-MS/MS was performed using an integrated system, which consisted of an autosampler switching pump and a micropump (Tempo Nano LC system; Applied Biosystems) with a hybrid quadrupole-TOF LC-MS/MS spectrometer (QStar Elite; Applied Biosystems) that was equipped with a nanoelectrospray ionization source (Applied Biosystems) and fitted with a 10
Peptides were first trapped on a Zorbax 300SB-C18 trap column (300
For data acquisition, the mass spectrometer was set in the positive ion mode at a selected mass range of 400–1600
Data file processing, protein identification, and relative abundance quantification were performed using ProteinPilot v.2.0.1 (Applied Biosystems; MDS-Sciex, Concord, Canada) and the Paragon algorithm [
The confidence threshold for protein identification was an unused ProtScore >1.3 (95% confidence interval). ProteinPilot computes a percentage confidence that reflects the probability that a hit is a false positive; thus, at the 95% confidence level, the false positive identification rate is approximately 5% [
The “biological process” and “molecular function” classifications were analyzed using PANTHER ID numbers (
Urine samples were pooled from 16 type 2 diabetic patients with normoalbuminuria and 16 type 2 diabetic patients with microalbuminuria. The characteristics of the pooled urine samples for 2-DE are shown in Figure
Twenty-four urine samples that were matched for gender and age (NA: 6 females and 6 males, and MA: 6 females; 6 males) were selected from the urine sample groups (NA1–NA4 and MA1–MA4, resp.) and subjected to Western blot validation of the 6 representative candidates from the iTRAQ experiments (Figures
In addition to Western blot, multiple reaction monitoring (MRM) was performed to verify the candidate biomarkers using 9 NA and 14 MA urine samples from the urine sample groups (NA1–NA4 and MA1–MA4, resp.) (Figure
For the iTRAQ experiments, 3 biological replicates (biological replicate 1 was labeled (a), replicate 2 was labeled (d), and replicate 3 was labeled (e)); 2 technical replicates (technical replicate 1 was labeled (a) and (c), replicate 2 was labeled (b) and (d)) were generated from normoalbuminuric and microalbuminuric urine (Figure
Seven hundred ten proteins were identified from 21,610 peptides of the 3 combined biological replicates at a minimum confidence level of 95% (unused ProtScore > 1.3). Of the proteins that were identified by iTRAQ, 27% comprised 1-peptide proteins; 14% was 2-peptide proteins; 8% was 3-peptide proteins; 5% was 4-peptide proteins; 46% comprised proteins that had 5 or more peptides. In our iTRAQ experiment, 83 proteins (unused ProtScore > 1.3) were common to all 3 biological replicates at a minimum confidence level of 95%, using 3 different pooled urine samples.
To generate the quantitative proteome using iTRAQ labeling, we first determined the labeling efficiency, which exceeded 98% (data not shown). Next, the cutoff for significant fold-change was determined based on the 2 technical replicates ((a) and (c) of iTRAQ experiment 1, (b); (d) of iTRAQ experiment 2) (Figure
The technical variations for the 115/114 and 117/116 reporter ions, calculated using the ratios of the 44 and 26 commonly observed proteins between the 2 technical replicates, were
Correlation between the 2 technical replicates and determination of the cutoff value for significant fold changes. (a) Plots of iTRAQ ratios for two technical replicates. Forty-four proteins were commonly observed from technical replicate 1 (labeled 115/114), and 26 proteins were commonly observed from technical replicate 2 (labeled 117/116). These differentially excreted proteins (
In our iTRAQ study, we obtained diverse biomarker candidates from 3 pooled biological NA/MA urine samples (each pooled NA or MA urine sample consisted of 9 individual urine specimens; thus, the 3 pooled NA, and 3 pooled MA urine samples comprised 54 different individual urine samples). Further, biomarker candidates were confirmed and validated by 2-DE, Western blot, and MRM.
To analyze urinary proteomes in normoalbuminuria and microalbuminuria subjects, 3 biological replicates were generated, wherein 196 proteins met the following criteria:
These proteins were further analyzed by differential proteomic expression. All quantified proteins were classified into “biological process” and “molecular function” subcategories using the PANTHER classification program, allowing us to analyze phenotypic features and molecular functions between microalbuminuria and normoalbuminuria (Figure
Comprehensive functional annotation of the differentially excreted proteome. The 196 quantitated urinary proteins were annotated for the “biological process” (
The 196 proteins from the 3 biological replicates were categorized by PANTHER ID number into “biological process” and “molecular function” groups; certain subcategories are summarized in Figures
Functional distribution of differentially excreted proteins in microalbuminuria versus normoalbuminuria. Functional classification of differentially excreted proteins into (a) “biological process” and (b) “molecular function.” Only 5 major subcategories for “biological process” and “molecular function” are shown; each subcategory is presented as the percentage of up- and down-regulated proteins.
The “biological process” subcategories accounted for 196 differentially excreted proteins, wherein “immunity and defense” and “protein metabolism” represented 49 and 34 of the quantitated proteins, respectively—the 2 largest components (Figure
The “molecular function” subcategories accounted for 196 differentially excreted proteins, in which “immunity” and “receptor” represented 25 and 19 of the quantitated proteins, respectively—the 2 largest components (Figure
One hundred ninety-six tentative biomarker candidates were differentially expressed, based on the iTRAQ data, and they were characterized biologically according to “biological process” and “molecular function.” Moreover, in a detailed association study of diabetic nephropathy and differentially excreted proteins using references and databases, we prioritized 10 candidates, of the 196 differentially excreted proteins (Figure
Selected 10 differentially excreted proteins related to pathogenic status in microalbumiuric versus normoalbuminuric urines.
Pathogenic status | Number of unique peptidesa | Accession numberb | Gene namec | MA : NA expression | |||
---|---|---|---|---|---|---|---|
iTRAQd | 2-DEe | WBf | |||||
Glomerular dysfunction | 1 | 15 | spt∣P02787 | 1.86 | — | ||
2 | 47 | Spt∣P00450 | 2.09 | — | |||
3 | 209 | spt∣P01009 | 1.42 | — | |||
4 | 49 | spt∣P00738 | 2.35 | — | |||
5 | 10 | trm∣Q9UMV3 | 0.29 | — | |||
6 | 235 | spt∣P98160 | 0.68 | — | |||
Tubular dysfunction | 7 | 9 | spt∣P02774 | 2.44 | — | ||
8 | 414 | spt∣P02763 | 2.04 | — | |||
Other types of diseases | 9 | 9 | spt∣Q01469 | 0.29 | — | ||
10 | 5 | trm∣O43653 | 1.70 | — | — |
aThe numbers of unique peptides and MS/MS spectrum observed by ProteinPilot software were determined only for those peptides with ≥95% confidence. bAccession numbers represent entries in the Human CDS database (human KBMS 5.0, 2005-03-02; a total of 187,748 entries provided by Applied Biosystems). cGene name from the Expasy database correspond to protein accession number bfrom the Human CDS database (human KBMS 5.0, 2005-03-02; a total of 187,748 entries provided by Applied Biosystems). d–fRatio of differentially excreted protein for iTRAQ, 2-DE and WB in microalbumiuric versus normoalbuminuric urines, respectively.
In the 3 biological replicates, transferrin (
Moreover, several differentially excreted proteins that were related to tubular dysfunction, such as vitamin D-binding protein (
In addition,
Differential protein expression between microalbuminuric and normoalbuminuric urine was also measured using the 2-D gel electrophoresis in pooled NA4 and MA4 urine (Figure
Differentially excreted proteins by 2-DE in microalbuminuria versus normoalbuminuria. (a) Representative whole 2-DE images of normoalbuminuric (NA) and microalbuminuric (MA) urine. Total protein (100
Validation using Western blot for six representative differentially excreted proteins. The concentrations of transferrin (a), ceruloplasmin (b),
Of the 7 proteins that were identified by 2-DE, 4 had the same pattern of differential excretion as in the iTRAQ experiment. Specifically, the spots that corresponded to serum albumin were upregulated by 2-DE (Figure
Differentially expressed proteins by 2-DE in microalbuminuria versus normoalbuminuria.
Gene namea | Accession numberb | Up-/down-regulated | Mol. Mass, Da (pI)c | Peptides matched | Total ion C.I.%d | Ma/Na | Ma/Na (iTRAQ)g |
---|---|---|---|---|---|---|---|
P02768 | Up | 71317.2 (5.92) | 2 | 100.00 | 3.09 | ||
P02768 | Up | 71317.2 (5.92) | 2 | 99.84 | 3.09 | ||
P02768 | Up | 71317.2 (5.92) | 2 | 100.00 | 3.09 | ||
P98160 | Down | 468527.5 (6.06) | 2 | 100.00 | 0.68 | ||
Q01469 | Down | 15496.7 (6.6) | 1 | 97.76 | 0.29 | ||
Q9UMV3 | Down | 75684.6 (5.47) | 2 | 100.00 | 0.29 | ||
P02760 | Down | 38974 (5.95) | 2 | 99.91 | 1.44 | ||
Q9NZ71 | Up | 152278.2 (8.68) | 1 | 96.77 | — | ||
Q9UBX5 | Down | 50146.7 (4.58) | 1 | 99.36 | — |
a-bGene name from the Expasy database correspond to protein accession number. bAccession numbers represent entries in the Human CDS database. cMolecular mass (mol. mass) is presented by Da, while isoelectric point stands for pI. dTotal ion score and total ion CI % for MALDI-TOF/TOF were calculated using GPS v3.5 in the MASCOT search program (v2.0). f-gRatio of differentially excreted protein for 2-DE and iTRAQ in microalbumiuric versus normoalbuminuric urines, respectively. Data are expressed as the mean ± SD.
To validate the differentially excreted proteins from the iTRAQ results, 6 proteins (
To verify the 7 biomarker candidates (
Parameters of MRM Experiment for seven candidate proteins.
Protein name | Q1a | Q3b | Sequencec | Fragmentd | Chargee | CEf |
---|---|---|---|---|---|---|
Transferrin | 482.8 | 682.4 | APNHAVVTR | y6 | 2+ | 26 |
315.2 | 558.3 | AVGNLR | y5 | 2+ | 19 | |
315.2 | 459.3 | AVGNLR | y4 | 2+ | 19 | |
Ceruloplasmin | 686.4 | 1080.0 | GAYPLSIEPIGVR | y10 | 2+ | 35 |
686.39 | 870.5 | GAYPLSIEPIGVR | y8 | 2+ | 35 | |
Alpha-1-antitrypsin | 555.81 | 997.5 | LSITGTYDLK | y9 | 2+ | 29 |
555.81 | 797.4 | LSITGTYDLK | y7 | 2+ | 29 | |
393.2 | 587.3 | VVNPTQK | y5 | 2+ | 22 | |
393.2 | 473.3 | VVNPTQK | y5 | 2+ | 22 | |
Haptoglobin precursor | 729.8 | 1084.5 | NLFLNHSENATAK | y10 | 2+ | 37 |
352.2 | 517.3 | VSVNER | y4 | 2+ | 20 | |
Vitamin D-binding protein | 400.2 | 700.4 | VLEPTLK | y6 | 2+ | 26 |
400.2 | 587.3 | VLEPTLK | y5 | 2+ | 26 | |
Alpha-1-acid glycoprotein 1 | 556.8 | 811.4 | SDVVYTDWK | y6 | 2+ | 29 |
580.8 | 974.5 | WFYIASAFR | y8 | 2+ | 31 | |
580.8 | 827.4 | WFYIASAFR | y7 | 2+ | 31 | |
Prostate stem cell antigen | 501.0 | 830.5 | AVGLLTVISK | y8 | 2+ | 30 |
501.0 | 660.4 | AVGLLTVISK | y6 | 2+ | 30 |
a-bQ1 and Q3 (
MRM validation was assessed by interactive plots and ROC curves, represented by the peak area of each Q1/Q3 transition. Figure
ROC curves and interactive plots for MRM validation in normoalbuminuric versus microalbuminuric urine. Seven biomarker candidates (
ROC curves for three candidate biomarkers and the 3-marker panel. MRM validation was performed for (a) alpha-1-antitrypsin, (b) alpha-1-acid glycoprotein 1, (c) prostate stem cell antigen, and (d) their combination, generating AUC values of 0.849, 0.873, and 0.825, respectively, whereas the combination resulted in a merged AUC of 0.921.
In the interactive plots,
To identify and quantify proteins that were associated with diabetic nephropathy in microalbuminuric and normoalbuminuric urine, we used relative quantitative proteomic techniques, such as iTRAQ, 2-DE, Western blot, and MRM. In our iTRAQ experiment, 710 urinary proteins were identified at a >95% confidence level, of which 196 were differentially excreted by >1.25 or <0.80—99 and 97 proteins were up- and down-regulated, respectively (Appendix
Recently, the Urinary Protein Biomarker (UPB) database was constructed and published, in which 205 publications were curated manually [
The “molecular function” subcategories accounted for 196 differentially excreted proteins, of which “immunity” represented 25 of the quantitated proteins—the largest component (Figure
Consequently, 10 proteins were selected for preliminary validation studies such as 2-DE, Western blot, and MRM. For example, 3 proteins (
Recently, an optimized quantitative proteomic strategy in urinary proteomic analysis was proposed for urine biomarker discovery using a small set of samples [
Nevertheless, an advantage of our study was that we used a large collection of urine samples from 86 diabetic patients to perform 3 iTRAQ experiments, including 3 biological replicates and 2 technical replicates, resulting in more reliable statistical significance.
Glomerular dysfunction is caused by GBM thickening and mesangial expansion due to ECM accumulation [
Transferrin-to-creatinine and ceruloplasmin-to-creatinine ratios are known to reflect changes in renal hemodynamics, and these ratios are significantly higher microalbuminuric patient than normoalbuminuric patients [
Haptoglobin (
Low-molecular-weight proteins, such as
For the MRM experiments, we used a bacterial beta galactosidase peptide as the internal standard for relative quatitation [
In the interactive plots,
Furthermore, we performed a multiplex assay to improve AUC values with 3 biomarker candidates (alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen), obtaining a merged AUC value of 0.921, which is greater than those of the individual proteins (0.849, 0.873, and 0.825 for alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, and prostate stem cell antigen, resp.) (Figure
Although our results require further validation in a larger collection of urine samples that contains various control samples, it appears that
Microalbuminuria is used as a noninvasive index for the detection of diabetic renal disease. Yet, more specific and accurate biomarkers for DN are required, particularly in type 2 diabetic patients, due to several reasons, including nonspecific detection in nondiabetic renal disease, cardiovascular disease, inflammation, and hypertension. In our iTRAQ experiments, 710 urinary proteins were identified at a >95% confidence level, of which 196 were differentially excreted by >1.25 or <0.80—99 and 97 proteins were up- and down-regulated, respectively (Appendix
We prioritized 196 proteins to select preliminary biomarker candidates by characterizing them with regard to “biological process” and “molecular function” and associating them with pathogenesis. Consequently, 10 proteins were selected. To confirm and validate these candidates, 2-DE, Western blot, and MRM were performed. Based on the MRM results,
Further validation studies on other differentially excreted proteins might contribute to a greater understanding of the mechanism of renal dysfunction and its association with the pathogenesis of DN, facilitating the development of better biomarkers for DN.
For more details, see Table
Differentially excreted urinary proteome in microalbumiuric versus normoalbuminuric urine.
Unique peptidesa | Accession numberb | Protein name | Ratioc MA : NA | Pvald MA : NA | EFe MA : NA | |
---|---|---|---|---|---|---|
1 | 651 | spt∣P02768 | Serum albumin | 3.09 | 0.00 | 1.04 |
2 | 337 | gb∣AAF01333.1 | Serum albumin | 0.36 | 0.00 | 1.08 |
3 | 669 | trm∣Q8N4N0 | Alpha-2-glycoprotein 1 | 1.48 | 0.00 | 1.02 |
4 | 639 | rf∣NP_003352.1 | uromodulin | 0.72 | 0.00 | 1.04 |
5 | 269 | emb∣CAA42438.1 | Zn-alpha2-glycoprotein | 1.80 | 0.00 | 1.06 |
6 | 235 | spt∣P98160 | HSPG | 0.68 | 0.00 | 1.04 |
7 | 209 | spt∣P01009 | Alpha-1-antitrypsin | 1.42 | 0.00 | 1.04 |
8 | 414 | spt∣P02763 | Alpha-1-acid glycoprotein 1 | 2.04 | 0.00 | 1.03 |
9 | 265 | spt∣P02788 | Serotransferrin | 2.46 | 0.00 | 1.10 |
10 | 46 | spt∣P02760 | AMBP protein | 1.44 | 0.00 | 1.12 |
11 | 300 | spt∣P07911 | Uromodulin | 0.24 | 0.00 | 1.08 |
12 | 106 | prf∣765044A | Ig G1 H Nie | 0.61 | 0.00 | 1.15 |
13 | 223 | dbj∣BAC85395.1 | Unnamed protein product | 1.37 | 0.00 | 1.10 |
14 | 217 | emb∣CAA29229.1 | Alpha-1-acid glycoprotein 1 | 2.29 | 0.00 | 1.11 |
15 | 107 | trm∣Q5VU27 | Heparan sulfate proteoglycan 2 | 2.00 | 0.00 | 1.18 |
16 | 306 | spt∣P07998 | Ribonuclease pancreatic | 0.80 | 0.00 | 1.08 |
17 | 62 | spt∣P41222 | Prostaglandin-H2 D-isomerase | 1.40 | 0.00 | 1.13 |
18 | 47 | spt∣P00450 | Ceruloplasmin | 2.09 | 0.00 | 1.12 |
19 | 117 | prf∣763134A | Ig A1 Bur | 1.60 | 0.02 | 1.39 |
20 | 46 | cra∣hCP1909255 | Serine proteinase inhibitor | 1.52 | 0.00 | 1.07 |
21 | 90 | spt∣P10451 | Osteopontin | 0.57 | 0.00 | 1.43 |
22 | 9 | spt∣P04746 | Pancreatic alpha-amylase | 0.41 | 0.00 | 1.33 |
23 | 39 | spt∣P02749 | Beta-2-glycoprotein I | 1.37 | 0.00 | 1.07 |
24 | 50 | trm∣Q9UII8 | E-cadherin | 1.36 | 0.00 | 1.12 |
25 | 34 | rf∣NP_006112.2 | Keratin 1 | 0.60 | 0.00 | 1.16 |
26 | 88 | spt∣Q14624 | ITIH4 | 0.78 | 0.00 | 1.08 |
27 | 21 | trm∣Q6N025 | FN | 1.27 | 0.01 | 1.20 |
28 | 21 | trm∣Q8N175 | Keratin 10 | 0.67 | 0.00 | 1.27 |
29 | 34 | emb∣CAA48671.1 | Alpha1-antichymotrypsin | 1.64 | 0.00 | 1.17 |
30 | 50 | spt∣P00738 | Haptoglobin | 2.36 | 0.01 | 1.24 |
31 | 22 | gb∣AAA52014.1 | Cholesterol esterase | 0.46 | 0.00 | 1.10 |
32 | 63 | spt∣P05451 | Lithostathine 1 alpha | 1.54 | 0.00 | 1.05 |
33 | 31 | trm∣Q6PAU9 | Kininogen 1 | 0.75 | 0.00 | 1.18 |
34 | 20 | spt∣P04217 | Alpha-1B-glycoprotein | 1.86 | 0.00 | 1.31 |
35 | 34 | spt∣P05155 | Plasma protease C1 inhibitor | 0.75 | 0.00 | 1.12 |
36 | 14 | trm∣Q8N473 | Alpha 1 type I collagen | 0.72 | 0.00 | 1.14 |
37 | 139 | trm∣Q6IB74 | ORM2 protein | 1.48 | 0.00 | 1.23 |
38 | 22 | spt∣Q8WZ75 | Roundabout homolog 4 | 0.34 | 0.00 | 1.23 |
39 | 29 | spt∣P02791 | Hemopexin | 1.73 | 0.01 | 1.33 |
40 | 33 | trm∣Q6LBL5 | GM2 activator protein | 1.45 | 0.00 | 1.06 |
41 | 19 | pdb∣1HP7_A | A Chain A, uncleaved alpha-1-antitrypsin | 1.76 | 0.00 | 1.18 |
42 | 23 | spt∣P55290 | Cadherin-13 | 0.76 | 0.00 | 1.13 |
43 | 21 | trm∣Q8IZY7 | Poly-Ig receptor | 0.63 | 0.00 | 1.36 |
44 | 25 | spt∣P05154 | Plasma serine protease inhibitor | 0.76 | 0.00 | 1.12 |
45 | 10 | trm∣Q96CZ9 | Cadherin 11, type 2, isoform 1 preproprotein | 0.44 | 0.00 | 1.50 |
46 | 16 | gb∣AAR84237.2 | Truncated epidermal growth factor | 0.48 | 0.00 | 1.25 |
47 | 35 | spt∣P24855 | Deoxyribonuclease I | 0.50 | 0.00 | 1.12 |
48 | 17 | trm∣Q7Z645 | Collagen, type VI, alpha 1 | 0.51 | 0.00 | 1.17 |
49 | 15 | dbj∣BAA19556.1 | Immunoglobulin light chain V-J region | 1.69 | 0.01 | 1.38 |
50 | 33 | emb∣CAA23842.1 | Unnamed protein product | 1.43 | 0.00 | 1.05 |
51 | 32 | spt∣P08571 | CD14 | 2.36 | 0.00 | 1.62 |
52 | 26 | trm∣Q6GMX2 | Hypothetical protein | 0.59 | 0.01 | 1.24 |
53 | 111 | emb∣CAA29873.2 | Alpha-1-acid glycoprotein 2 | 2.25 | 0.00 | 1.18 |
54 | 7 | trm∣Q8WY99 | Cathepsin C | 1.58 | 0.00 | 1.17 |
55 | 11 | spt∣Q92820 | Gamma-glutamyl hydrolase | 0.54 | 0.01 | 1.33 |
56 | 13 | spt∣P15586 | N-acetylglucosamine-6-sulfatase | 1.30 | 0.02 | 1.23 |
57 | 10 | gb∣AAQ88523.1 | AQGV3103 | 0.79 | 0.05 | 1.26 |
58 | 8 | trm∣Q8N2F4 | Hypothetical protein PSEC0200 | 0.71 | 0.01 | 1.29 |
59 | 20 | trm∣Q6MZU6 | Hypothetical protein DKFZp686C15213 | 0.39 | 0.00 | 1.28 |
60 | 27 | trm∣Q6LDS3 | APS protein | 1.34 | 0.00 | 1.08 |
61 | 9 | pdb∣1L9X_D | Structure Of Gamma-Glutamyl Hydrolase | 0.69 | 0.00 | 1.12 |
62 | 15 | spt∣P07339 | Cathepsin D | 1.38 | 0.01 | 1.26 |
63 | 10 | spt∣P51884 | Lumican | 0.78 | 0.01 | 1.20 |
64 | 130 | dbj∣BAC85483.1 | Unnamed protein product | 0.73 | 0.00 | 1.14 |
65 | 9 | pdb∣1ATH_B | B Chain B, Antithrombin Iii | 1.29 | 0.00 | 1.17 |
66 | 15 | rf∣NP_001822.2 | Clusterin isoform 1 | 0.63 | 0.00 | 1.25 |
67 | 23 | trm∣Q5VW91 | Decay accelerating factor for complement | 1.26 | 0.00 | 1.09 |
68 | 7 | spt∣P54802 | Alpha-N-acetylglucosaminidase | 0.50 | 0.00 | 1.25 |
69 | 12 | spt∣Q16270 | IGFBP-7 | 0.79 | 0.00 | 1.14 |
70 | 8 | trm∣Q5VZE3 | Golgi phosphoprotein 2 | 0.38 | 0.02 | 1.46 |
71 | 10 | spt∣P05543 | Thyroxine-binding globulin | 1.27 | 0.00 | 1.11 |
72 | 9 | spt∣P02774 | Vitamin D-binding protein | 2.44 | 0.00 | 1.15 |
73 | 63 | rf∣NP_000573.1 | Secreted phosphoprotein 1 | 0.58 | 0.00 | 1.24 |
74 | 56 | spt∣P02671 | Fibrinogen alpha/alpha-E chain | 0.67 | 0.00 | 1.11 |
75 | 10 | trm∣Q9UBG3 | Tumor-related protein | 0.32 | 0.00 | 1.67 |
76 | 15 | trm∣O00391 | Quiescin Q6 | 0.77 | 0.03 | 1.25 |
77 | 36 | trm∣Q5VY30 | Retinol binding protein 4, plasma | 1.38 | 0.00 | 1.04 |
78 | 8 | rf∣NP_004675.2 | SPARC-like 1 | 0.55 | 0.03 | 1.70 |
79 | 9 | spt∣Q92692 | Herpesvirus entry mediator B | 0.50 | 0.00 | 1.31 |
80 | 39 | trm∣Q96FE7 | HGFL protein | 0.73 | 0.00 | 1.15 |
81 | 6 | spt∣P43652 | Afamin | 4.67 | 0.00 | 1.33 |
82 | 24 | pdb∣1QDD_A | Lithostathine | 1.73 | 0.00 | 1.20 |
83 | 24 | spt∣P10153 | Nonsecretory ribonuclease | 0.83 | 0.01 | 1.13 |
84 | 8 | spt∣P16278 | Beta-galactosidase | 1.50 | 0.00 | 1.20 |
85 | 7 | trm∣Q5VYK1 | Collagen, type XII, alpha 1 | 0.75 | 0.00 | 1.15 |
86 | 9 | emb∣CAA37914.1 | Precursor (AA-19 to 692) | 1.91 | 0.01 | 1.62 |
87 | 15 | trm∣Q7Z5L0 | Unnamed secretory protein | 0.56 | 0.00 | 1.31 |
88 | 11 | spt∣P08236 | Beta-glucuronidase | 1.33 | 0.00 | 1.12 |
89 | 13 | cra∣hCP51001.2 | superoxide dismutase 3 | 0.45 | 0.00 | 1.38 |
90 | 17 | pir∣S13195 | Ganglioside M2 activator protein | 1.33 | 0.00 | 1.18 |
91 | 12 | cra∣hCP1858145 | Protein C receptor, endothelial | 1.42 | 0.00 | 1.22 |
92 | 12 | trm∣Q6IAT8 | B2M protein | 1.48 | 0.00 | 1.08 |
93 | 7 | trm∣Q9Y5X6 | Glutamate carboxypeptidase | 1.47 | 0.00 | 1.04 |
94 | 13 | spt∣P06702 | Calgranulin B | 0.41 | 0.00 | 1.36 |
95 | 11 | emb∣CAB90482.1 | Human type XVIII collagen | 0.56 | 0.01 | 1.48 |
96 | 7 | trm∣O00533 | Neural cell adhesion molecule | 0.73 | 0.02 | 1.29 |
97 | 72 | spt∣P04745 | Salivary alpha-amylase | 1.25 | 0.00 | 1.12 |
98 | 40 | spt∣O75594 | Peptidoglycan recognition protein | 0.56 | 0.00 | 1.08 |
99 | 127 | emb∣CAA40946.1 | Immunoglobulin lambda light chain | 1.71 | 0.01 | 1.47 |
100 | 15 | trm∣Q9UJ36 | Transmembrane glycoprotein | 0.74 | 0.01 | 1.25 |
101 | 4 | gb∣AAH17802.1 | SPRR3 protein | 0.14 | 0.00 | 1.25 |
102 | 9 | spt∣Q01469 | Fatty acid-binding protein | 0.29 | 0.00 | 1.34 |
103 | 4 | trm∣Q6FGL5 | LCN2 protein | 1.36 | 0.01 | 1.33 |
104 | 10 | trm∣Q9UMV3 | MBL-associated serine protease 2 | 0.29 | 0.00 | 1.34 |
105 | 10 | gb∣AAH30653.1 | Cadherin 13, preproprotein | 2.91 | 0.00 | 1.76 |
106 | 6 | spt∣Q9H8L6 | Multimerin 2 | 0.69 | 0.02 | 1.34 |
107 | 33 | trm∣Q9UD19 | Intron-containing kallikrein | 0.73 | 0.02 | 1.30 |
108 | 4 | spt∣P07195 | L-lactate dehydrogenase B chain | 0.75 | 0.00 | 1.16 |
109 | 9 | spt∣P08185 | Corticosteroid-binding globulin | 3.22 | 0.00 | 1.79 |
110 | 14 | trm∣Q5UGI3 | Ubiquitin C splice variant | 1.37 | 0.01 | 1.24 |
111 | 6 | pdb∣1O1P_D | D Chain D, Deoxy Hemoglobin | 0.55 | 0.00 | 1.32 |
112 | 7 | spt∣P80723 | Brain acid soluble protein 1 | 2.24 | 0.00 | 1.46 |
113 | 7 | spt∣P19320 | Vascular cell adhesion protein 1 | 1.25 | 0.03 | 1.23 |
114 | 6 | spt∣P27797 | Calreticulin | 1.39 | 0.00 | 1.13 |
115 | 10 | spt∣Q01459 | Di-N-acetylchitobiase | 1.43 | 0.01 | 1.26 |
116 | 6 | trm∣Q6PN97 | Alpha 2 macroglobulin | 3.04 | 0.00 | 1.84 |
117 | 5 | gb∣AAV40827.1 | superoxide dismutase 3 | 0.48 | 0.00 | 1.15 |
118 | 3 | spt∣P08473 | Neprilysin | 2.26 | 0.00 | 1.51 |
119 | 61 | trm∣Q9Y5Y7 | LYVE-1 | 1.63 | 0.00 | 1.03 |
120 | 2 | spt∣P26038 | Moesin | 1.56 | 0.00 | 1.18 |
121 | 9 | trm∣Q6PIJ0 | FCGR3A protein | 0.77 | 0.00 | 1.06 |
122 | 7 | spt∣P14209 | T cell surface glycoprotein E2 | 2.02 | 0.02 | 1.61 |
123 | 6 | spt∣P02765 | Alpha-2-HS-glycoprotein | 1.69 | 0.00 | 1.20 |
124 | 215 | pir∣A23746 | Ig kappa chain V-III | 1.31 | 0.00 | 1.14 |
125 | 3 | trm∣Q9NT71 | Hypothetical protein DKFZp761A051 | 0.74 | 0.03 | 1.31 |
126 | 6 | trm∣Q9Y4W4 | Type XV collagen | 0.52 | 0.00 | 1.25 |
127 | 5 | cra∣hCP42501.1 | Complement component 1 | 0.74 | 0.00 | 1.20 |
128 | 11 | spt∣P09564 | T-cell antigen CD7 | 0.67 | 0.01 | 1.26 |
129 | 6 | dbj∣BAA86053.1 | Carboxypeptidase E | 0.79 | 0.02 | 1.20 |
130 | 6 | spt∣P15151 | Poliovirus receptor | 1.46 | 0.01 | 1.51 |
131 | 13 | trm∣Q8IUP2 | Protocadherin 1, isoform 1 | 0.49 | 0.02 | 1.75 |
132 | 3 | trm∣Q8NBK0 | Hypothetical protein | 1.31 | 0.01 | 1.19 |
133 | 5 | spt∣P22891 | Vitamin K-dependent protein Z | 0.76 | 0.01 | 1.21 |
134 | 6 | trm∣Q9UNF4 | Hyaluronic acid receptor | 1.50 | 0.01 | 1.29 |
135 | 11 | trm∣Q9HCU0 | Tumor endothelial marker 1 | 0.46 | 0.00 | 1.36 |
136 | 8 | spt∣P35527 | Keratin, type I cytoskeletal 9 | 0.55 | 0.00 | 1.29 |
137 | 3 | spt∣P55285 | Cadherin-6 | 0.41 | 0.00 | 1.39 |
138 | 3 | trm∣Q9BYH7 | Scavenger receptor with C-type lectin type I | 1.39 | 0.01 | 1.23 |
139 | 2 | trm∣Q13942 | Calmodulin | 2.64 | 0.00 | 1.39 |
140 | 8 | spt∣P04004 | Vitronectin | 1.87 | 0.00 | 1.42 |
141 | 11 | trm∣Q86Z23 | Hypothetical protein | 2.11 | 0.01 | 1.49 |
142 | 140 | trm∣Q9NWE3 | Hypothetical protein FLJ10084 | 1.55 | 0.00 | 1.04 |
143 | 99 | trm∣Q9NWE3 | Hypothetical protein FLJ10084 | 1.53 | 0.00 | 1.06 |
144 | 11 | spt∣P05109 | Calgranulin A | 0.42 | 0.00 | 1.34 |
145 | 58 | trm∣Q9NWE3 | Hypothetical protein FLJ10084 | 1.50 | 0.00 | 1.08 |
146 | 3 | trm∣Q9BYH7 | Scavenger receptor with C-type lectin type I | 1.65 | 0.00 | 1.24 |
147 | 4 | gb∣AAA52018.1 | Chromogranin A | 0.52 | 0.00 | 1.26 |
148 | 4 | emb∣CAI20248.1 | PPGB | 1.51 | 0.00 | 1.24 |
149 | 92 | pir∣S12443 | Ig lambda chain (Ke+O−)−human | 1.35 | 0.03 | 1.24 |
150 | 7 | trm∣Q5TEQ5 | OTTHUMP00000044363 | 0.29 | 0.00 | 1.58 |
151 | 18 | trm∣Q6UX86 | GPPS559 | 2.08 | 0.01 | 1.60 |
152 | 2 | trm∣Q8TCZ2 | MIC2L1 | 0.80 | 0.01 | 1.16 |
153 | 5 | spt∣P61970 | Nuclear transport factor 2 | 0.78 | 0.01 | 1.18 |
154 | 284 | pdb∣1T04_C | Anti-ifn-gamma fab in C2 space group | 1.45 | 0.02 | 1.28 |
155 | 8 | spt∣P00790 | Pepsin A | 0.80 | 0.00 | 1.15 |
156 | 2 | cra∣hCP1778903.1 | CD7 antigen | 0.48 | 0.00 | 1.03 |
157 | 5 | trm∣O00480 | Butyrophilin, subfamily 2 | 0.68 | 0.00 | 1.12 |
158 | 5 | trm∣O43653 | Prostate stem cell A | 1.70 | 0.00 | 1.25 |
159 | 4 | trm∣Q9BX83 | Hemoglobin alpha 1 globin chain | 0.67 | 0.05 | 1.47 |
160 | 9 | spt∣P11684 | Uteroglobin | 3.16 | 0.00 | 1.86 |
161 | 7 | trm∣Q7LDY7 | Alpha-KG-E2 | 1.47 | 0.00 | 1.17 |
162 | 3 | trm∣Q9BYH7 | Scavenger receptor | 1.92 | 0.00 | 1.25 |
163 | 4 | trm∣Q6AZK5 | KRT13 protein | 0.56 | 0.02 | 1.58 |
164 | 5 | spt∣P09619 | PDGF-R-beta | 0.66 | 0.01 | 1.29 |
165 | 5 | pdb∣5TTR_H | Leu 55 Pro Transthyretin | 1.46 | 0.02 | 1.33 |
166 | 3 | rf∣NP_877418.1 | Mucin 1, transmembrane | 0.49 | 0.02 | 1.78 |
167 | 2 | trm∣Q5SWW9 | OTTHUMP00000060590 | 0.39 | 0.01 | 1.76 |
168 | 2 | rf∣NP_003217.2 | Trefoil factor 3 | 1.63 | 0.00 | 1.22 |
169 | 3 | spt∣Q99574 | Neuroserpin | 0.63 | 0.01 | 1.36 |
170 | 21 | trm∣Q9Y3U9 | Hypothetical protein DKFZp566C243 | 0.75 | 0.00 | 1.06 |
171 | 23 | gb∣AAB27607.1 | Prostaglandin D synthase | 1.20 | 0.00 | 1.10 |
172 | 4 | gb∣AAO11857.1 | Immunoglobulin | 1.38 | 0.00 | 1.14 |
173 | 2 | trm∣Q5VTA6 | Cubilin | 0.79 | 0.03 | 1.23 |
174 | 3 | trm∣Q5SY67 | OTTHUMP00000059857 | 0.69 | 0.02 | 1.33 |
175 | 54 | spt∣P15814 | Immunoglobulin lambda-like polypeptide 1 | 2.50 | 0.01 | 1.37 |
176 | 2 | spt∣P22352 | Plasma glutathione peroxidase | 0.70 | 0.00 | 1.13 |
177 | 7 | gb∣AAL68978.1 | Mutant beta globin | 0.32 | 0.00 | 1.77 |
178 | 6 | spt∣P35908 | KCytokeratin 2e | 0.50 | 0.03 | 1.74 |
179 | 284 | dbj∣BAB18261.1 | Anti-HBs antibody light chain | 3.19 | 0.00 | 1.53 |
180 | 3 | rf∣XP_370615.2 | Hypothetical protein | 0.53 | 0.03 | 1.72 |
181 | 164 | gb∣AAB50880.2 | Anitubulin IgG1 kappa VL chain | 1.64 | 0.00 | 1.30 |
182 | 170 | dbj∣BAC01692.1 | Immunoglobulin kappa light chain | 2.49 | 0.01 | 1.79 |
183 | 2 | trm∣Q7RTN9 | Type II keratin K6h | 0.43 | 0.01 | 1.52 |
184 | 3 | spt∣P21926 | Motility-related protein | 1.70 | 0.03 | 1.45 |
185 | 2 | spt∣Q13873 | Bone morphogenetic protein receptor | 0.61 | 0.01 | 1.08 |
186 | 4 | gb∣AAA62175.1 | Heat shock protein 27 | 0.53 | 0.00 | 1.38 |
187 | 15 | spt∣P02787 | transferrin | 1.86 | 0.00 | 1.25 |
188 | 2 | spt∣P07108 | Acyl-CoA-binding protein | 2.20 | 0.02 | 1.47 |
189 | 2 | spt∣Q9NZH0 | G protein-coupled receptor family C | 1.27 | 0.04 | 1.25 |
190 | 2 | trm∣Q96E46 | Fructose-1,6-bisphosphatase 1 | 0.44 | 0.00 | 1.01 |
191 | 44 | gb∣AAB53267.1 | Immunoglobulin V-region light chain | 1.40 | 0.03 | 1.30 |
192 | 8 | gb∣AAR32503.1 | Immunoglobulin heavy chain | 0.46 | 0.00 | 1.19 |
193 | 7 | rf∣NP_653247.1 | Immunoglobulin J chain | 0.37 | 0.01 | 1.88 |
194 | 29 | emb∣CAA12585.1 | Ig heavy chain variable region | 1.50 | 0.00 | 1.13 |
195 | 4 | gb∣AAD16731.1 | Immunoglobulin lambda light chain | 1.58 | 0.00 | 1.11 |
196 | 33 | trm∣Q6UXB8 | HGSC289 (OTTHUMP00000039678) | 1.27 | 0.04 | 1.22 |
aThe numbers of unique peptides and MS/MS spectrum observed by ProteinPilot software were determined only for those peptides with ≥95% confidence. bAccession numbers represent entries in the Human CDS database (human KBMS 5.0, 2005-03-02; a total of 187,748 entries provided by Applied Biosystems). c–eThe iTRAQ ratio,
To verify the MRM validation, we analyzed minimum of 2 transitions for each biomarker candidate protein.
Results from Mascot search and ROC curves for each transition are summarized in Figures
J. Jin and Y. H. Ku contributed equally to this study.
This paper was supported by the 21C Frontier Functional Proteomics Project of the Korean Ministry of Science and Technology (Grant no. FPR 08-A2-110) and a Grant (no. 10035353) from the Seoul R&BD Program.