Conceived and designed the experiments: PY. Performed the experiments: LM. Analyzed the data: LM SH. Contributed reagents/materials/analysis tools: FL. Wrote the paper: LM PY AK.
The authors have declared that no competing interests exist.
Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4+ T cells have been shown to be involved in autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4+ T cell from patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the two groups. The results revealed that the expression of 102 peptides was significantly altered (p<0.05) between two groups and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA suggests a role for both proteins in the pathogenesis of this syndrome.
Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disorder mainly affecting systemic melanocytes including those in the eyes, meninges, ears, skin, and hair
Proteomics provides important tools for identifying molecules involved in both normal and pathological processes
Ten active VKH patients and eleven healthy individuals were included in the present study. CD4+ T cells from five active VKH patients and five healthy individuals were used for label-free proteomics analysis. CD4+ T cells from another group of five active VKH patients and six controls were used for a validation study.The diagnosis of VKH syndrome was made according to the criteria revised for VKH syndrome by an international nomenclature committee
Patient number | Sex | Age | Ocular Manifestation | Visual Acuity | Extraocular Findings | |||||||||
Mutton-KPs | Aqueous cells | Iris nodules | posterior synechia | Sunset glow fundus | Multiple peripheral chorioretinal lesions | od | os | Meningismus | Tinitus | Alopecia and poliosis | Vitiligo | |||
Screening | ||||||||||||||
1 | Male | 33 | ++ | +++ | present | present | 0.25 | 0.1 | + | - | + | + | ||
2 | Male | 34 | ++++ | ++ | present | present | 0.5 | 0.5 | + | + | + | - | ||
3 | Male | 39 | ++ | ++ | present | present | present | 0.08 | 0.06 | - | + | + | + | |
4 | Male | 24 | +++ | ++ | present | present | present | present | 0.06 | 0.05 | + | + | + | - |
5 | Female | 34 | ++ | ++ | present | present | present | 0.8 | 1.0 | - | + | + | + | |
Validation | ||||||||||||||
6 | Male | 18 | ++ | ++ | present | present | 0.7 | 0.6 | - | + | + | - | ||
7 | Female | 27 | ++ | +++ | present | present | present | 0.6 | 0.01 | + | - | + | - | |
8 | Female | 50 | ++ | ++ | present | present | present | 0.3 | 0.05 | + | + | + | - | |
9 | Male | 46 | ++ | +++ | present | present | 1.0 | 0.8 | - | + | + | + | ||
10 | Male | 41 | +++ | ++ | present | present | present | present | 0.2 | 0.5 | + | + | + | + |
The PBMCs were prepared from heparinized blood by Ficoll-Hypaque density-gradient centrifugation and were rinsed for three times in phosphate-buffered saline (PBS). Peripheral CD4+ T cells were purified using human CD4 microbeads according to the manufacturer's instructions (Miltenyi Biotec, Palo Alto, Calif). Briefly, PBMCs were suspended in 80 µl of PBS containing 0.5% bovine serum albumin (BSA) and 2 mM ethylenediamine tetraacetic acid (EDTA) per 107 total cells. A volume of 20 µl of CD4 microbeads was added to this suspension and incubation was performed for 15 min at 4°C. The cells were then washed in 2 ml of PBS containing 0.5% BSA and 2 mM EDTA and applied to a magnetic column on a MiniMACS separation unit (Miltenyi Biotec, Palo Alto, Calif). The CD4+ T cell fraction was collected and used in subsequent experiments. For flow cytometric analysis, aliquots of 1×106 PBMCs and isolated CD4+ T cells were stained with PE-cy7-conjugated monoclonal antibody (mAb) against human CD4 and appropriate isotype controls (eBioscience, San Diego, CA, USA) for 30 min at 4°C in the dark. Flow cytometric analysis was performed using FACS Calibur and CellQuest software (BD Biosciences, SanJose, CA).
Membrane proteins were extracted using a membrane protein extraction kit (Merck KGaA, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, 5×106 CD4+ T cells were pelleted by centrifugation at 300 g for five min. The supernatant was carefully removed and discarded. Reagent A (150 µl) was added to the cell pellet and a homogeneous cell suspension was prepared by pipetting up and down and was then incubated for 10 min at room temperature with occasional vortexing. The lysed cells were placed on ice. One part of reagent B with two parts of reagent C were mixed. A total amount of 450 µl of the mixed reagent B and C was added to each tube of lysed cells and then vortexed. The tubes were incubated on ice for 30 min with occasional vortexing. After centrifuging at 10,000 g for 3 min at 4°C, the supernatant was transferred to a new tube and was incubated for 10 min at 37°C to separate the membrane protein fraction. The tubes were then centrifuged at room temperature for 2 min at 10,000 g to isolate the hydrophobic fraction from the hydrophilic fraction. The hydrophilic phase (top layer) was removed carefully from the membrane-enriched phase (bottom layer). The protein concentration of the hydrophobic protein was determined using the protein assay kit (Bio-Rad, Hercules, CA). The protein was aliquoted and stored at −80°C until use.
Protein digestion was performed as follows. The protein concentration was adjusted to 5 ug/ul with lysis buffer. The aforementioned membrane protein was chemically reduced for 2.5 h at room temperature by adding DTT to 10 mM, and then carboxyamidomethylated in 50 mM iodoacetamide for 40 min at room temperature in the dark. Endoprotease LysC (Roche, Indianapolis, IN) was added to a final substrate: enzyme ratio of 100∶1 (w/w), and the reaction was incubated at 37° for 3 h. The urea concentration in protein samples was adjusted to 1.5 M with 25 mM NH4HCO3, and then 2 µg of trypsin was added to a final substrate: enzyme ratio of 50∶1 (w/w). The trypsin digest was incubated at 37° for 20 h. The peptide mixture was acidified by formic acid to 0.1% for further MS analysis.
Separation of the trypsin-digested peptides was performed with the Ettan MDLC system (GE Healthcare, Piscataway, NJ). Peptide samples were first desalted through a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) and then separated by a 0.15 mm*150 mm (RP-C18) column (Column Technology Inc, Fremont, CA) with micro spray mode. An aqueous 0.1% formic acid solution was used as phase A and a solution of 0.1% formic acid and 84% acetonitrile was used as phase B. The peptides were separated with a linear gradient of 4%∼50% mobile phase B over 2 hours at a flow rate of 1300 nl/min. The column temperature was maintained at 170 °C.Both pooled samples were analyzed in triplicate.
The separated peptides were analyzed on a Finnigan LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with a nanoelectrospray ion source
The peaklist of peptides was generated using DeCyder MS software version 1.0 (GE Healthcare, Piscataway, NJ) and the quantitative analysis of peptides was performed. Peptide detection, elution profile comparison, background subtraction and peptide quantitation were carried out on the full scan precursor mass spectra in fully automatic mode. Peptide quantitation is based on MS signal intensities of individual LC-MS analyses. Different signal intensity maps were matched using the pepmatch module and then the peptide quantitative results were acquired. As there was no internal standard, the intensity distributions for all peptides detected in both samples were used for normalization. Throughout these studies the mass tolerance in the software was set to 0.5 atomic mass unit and the retention time tolerance was set to 2 min.
Total protein of CD4+ T cells from five active VKH patients and six healthy individuals were prepared for Western blot analysis. Ten micrograms of protein from each specimen were used for SDS-PAGE. The gels were then transferred to a PVDF membrane. Membranes were incubated with antibodies at dilutions of 1∶1000 for both anti-human CD18 mAb (R&D systems, Minneapolis, MN) and anti-human AKNA mAb (Genway Biotech, San Diego, CA). Proteins were detected using the Phototope-HRP western blot detection system (Cell Signaling, Danvers, MA).
For differential analysis of peptides from both VKH patients and normal controls, student's
CD4+ T cells were sorted from peripheral blood of ten active VKH patients and eleven healthy individuals using CD4 microbeads. The purity of CD4+ T cells identified in both patients and controls was 98% (
The PBMC and freshly isolated CD4+ T cells were stained with the indicated markers using fluorescence-labeled mAb and analyzed by flow cytometry. Before sorting, the ratio of CD4+ T cells in PBMC was 38.11% (A). After microbeads based sorting, the purity of CD4+ T cells was as high as 98.06% (B).
Before conducting the relative protein profiling analysis between the two pooled samples from VKH patients and normal controls, several quality control measures were performed on the replicates of both pools to determine analytical reproducibility. The mass precision of the extracted peptide components was within 5 parts per million (ppm) of mass error. The quality control analysis revealed similar results between patients and controls concerning the median and average mass errors (1.90 vs 2.80 ppm), intensity errors (2.10% vs 1.95%), and retention time (0.70% vs 0.50%).
Reproducibility between duplicate runs was evaluated using a binary comparison map. The horizontal (x) axis represents the distribution of peak intensities of the first run, and the vertical (y) axis represents that of the second run. The average intensity correlation coefficient (CC) between the two runs was 0.92. A representative binary comparison map is shown in
The digested membrane proteins of CD4+ T cells prepared from active VKH patients and normal controls were analyzed by LC-MS/MS. Base peak ion chromatograms were acquired in profile mode to get enough data points for evaluation by DeCyder MS. A representative base peak ion chromatogram from the analysis of the VKH pool is shown in
The location of peptide ALNEITESGR in both samples was labeled with square frames in the total graphs and the magnified graphs (C and D). Statistical analysis showed that the different expression of peptide ALNEITESGR was significant between VKH group and normal group (E).
In total 354 peptides were identified to be significantly different between both groups, in which 102 could be sequenced and which corresponded to 64 proteins. A FDR of 3.77% at the peptide level was obtained by searching against the sequence-reversed decoy IPI human database, suggesting a high fidelity of this strategy. Thirty proteins showed a difference of at least 1.5-fold, when comparing active VKH patients with normal controls. Of these, eight proteins were identified based on two or more unique peptides (
Gene symbol | Swiss-prot accession No. | Identified proteins | Expression Ratio |
Xcorr |
t-test p | MH+ (Da) | Mass stdev (Da) | Identified Peptide |
VIM | B0YJC4 | Vimentin | 2.8 | 2.42 | 0.00004 | 1060.7690 | 0.08313 | 159 R.QVDQLTNDK.A 169 |
5.53 | 0.00002 | 1169.9804 | 0.11453 | 129 K.ILLAELEQLK.G 140 | ||||
2.27 | 0.00061 | 1428.8390 | 0.13478 | 50 R.SLYASSPGGVYATR.S 65 | ||||
3.16 | 0.00031 | 1115.8539 | 0.14490 | 105 K.VELQELNDR.F 115 | ||||
3.36 | 0.00032 | 1323.8875 | 0.13155 | 196 R.EEAENTLQSFR.Q 208 | ||||
3.99 | 0.00045 | 1406.1375 | 0.30727 | 223 K.VESLQEEIAFLK.K 236 | ||||
3.30 | 0.00030 | 1533.9759 | 0.26803 | 222 R.KVESLQEEIAFLK.K 236 | ||||
3.30 | 0.00029 | 1312.8985 | 0.35813 | 207 R.QDVDNASLAR.L 218 | ||||
2.26 | 0.00061 | 1226.9591 | 0.20369 | 294 K.FADLSEAANR.N 305 | ||||
KRT10 | P13645 | Keratin, type I cytoskeletal 10 | 2.0 | 2.26 | 0.00449 | 993.8803 | 0.14940 | 237 K.YENEVALR.Q 246 |
2.49 | 0.00409 | 1435.8816 | 1.01701 | 439 K.IRLENEIQTYR.S 451 | ||||
3.40 | 0.00108 | 2083.0051 | 0.17698 | 422 R.AETECQNTEYQQLLDIK.I 440 | ||||
2.27 | 0.02983 | 1262.8610 | 0.14280 | 450 R.SLLEGEGSSGGGGR.G 465 | ||||
3.03 | 0.00403 | 1031.5372 | 0.57250 | 257 R.VLDELTLTK.A 267 | ||||
3.84 | 0.00012 | 1494.0186 | 0.19081 | 322 R.SQYEQLAEQNRK.D 335 | ||||
2.26 | 0.00448 | 1236.916 | 0.16415 | 165 R.ALEESNYELEGK.I 178 | ||||
ATP5B | P06576 | ATP synthase subunit beta, mitochondrial precursor | 1.8 | 2.26 | 0.00433 | 1988.1477 | 0.17359 | 387 R.AIAELGIYPAVDPLDSTSR.I 407 |
3.57 | 0.00622 | 975.7282 | 0.15882 | 201 K.IGLFGGAGVGK.T 213 | ||||
2.67 | 0.00022 | 1088.6165 | 0.50286 | 188 K.VVDLLAPYAK.G 199 | ||||
ACTA2 | P62736 | Actin, aortic smooth muscle | 1.8 | 3.83 | 0.01604 | 1354.8744 | 0.12513 | 52 K.DSYVGDEAQSKR.G 65 |
3.12 | 0.00290 | 1198.7264 | 0.08676 | 52 K.DSYVGDEAQSK.R 64 | ||||
KRT2 | P62736 | Keratin, type II cytoskeletal 2 epidermal | 1.8 | 4.91 | 0.00099 | 1191.8102 | 0.09092 | 374 K.YEELQVTVGR.H 385 |
4.89 | 0.00012 | 1461.0514 | 0.45310 | 302 K.VDLLNQEIEFLK.V 315 | ||||
3.95 | 0.00025 | 1372.6575 | 0.48309 | 441 K.LNDLEEALQQAK.E 454 | ||||
KRT1 | P04264 | Keratin, type II cytoskeletal 1 | 1.6 | 2.24 | 0.00899 | 993.8803 | 0.14940 | 443 K.LNDLEDALQQAK.E 456 |
4.70 | 0.00027 | 1435.8816 | 1.01701 | 343 R.SLDLDSIIAEVK.A 356 | ||||
3.08 | 0.00072 | 2083.0051 | 0.17698 | 185 K.SLNNQFASFIDK.V 198 | ||||
ALB | A6NBZ8 | Uncharacterized protein ALB | 1.6 | 2.95 | 0.04948 | 1444.1611 | 0.54524 | 286 K.YICENQDSISSK.L 299 |
3.08 | 0.00704 | 1512.1173 | 0.04618 | 438 K.VPQVSTPTLVEVSR.N 453 | ||||
2.95 | 0.04947 | 1078.9591 | 0.10821 | 499 K.CCTESLVNR.R 509 | ||||
SFRS1 | Q07955 | Isoform ASF-1 of Splicing factor, arginine/serine-rich 1 | 0.50 | 2.52 | 0.00007 | 1255.5932 | 0.37017 | 17 R.IYVGNLPPDIR.T 29 |
2.78 | 0.03060 | 1078.9591 | 0.10822 | 154 R.DGTGVVEFVR.K 165 | ||||
3.37 | 0.00126 | 1417.7458 | 0.11825 | 142 R.EAGDVCYADVYR.D 155 |
a: average expression ratio;
b: cross-correlation score.
Genesymbol | Swiss-prot accession No. | Identified proteins | Expression Ratio |
Xcorr |
t-test p | MH+(Da) | Mass stdev(Da) | Identified Peptide |
ITGB2 (CD18) | A8MYE6 | Integrin beta | 2.9 | 3.66 | 0.00068 | 1047.7857 | 0.11764 | 520 R.TTEGCLNPR.R 530 |
ODZ3 | Q9P273 | Teneurin-3 | 2.5 | 4.03 | 0.00004 | 2254.3396 | 0.17867 | 897 R.QDGMFDLVANGGASLTLVFER.S 920 |
CORO2A | Q92828 | Coronin-2A | 2.4 | 2.21 | 0.00128 | 1550.6224 | 0.22761 | 235 K.KLMSTGTSRWNNR.Q 249 |
C19orf2 | Q8TC23 | C19orf2 protein | 2.3 | 2.91 | 0.01076 | 1088.1358 | 0.47804 | 344 R.INTGKNTTLK.F 355 |
DMD | P11532 | Isoform 4 of Dystrophin | 2.1 | 4.05 | 0.00033 | 1226.9591 | 0.20370 | 602 K.LAVLKADLEKK.K 614 |
SYCP2 | Q9BX26 | Synaptonemal complex protein 2 | 2.1 | 3.18 | 0.00454 | 1427.8801 | 0.06260 | 509 R.IKPPLQMTSSAEK.P 523 |
DKFZp686D0972 | Q562R1 | hypothetical protein LOC345651 | 1.9 | 2.27 | 0.00140 | 1791.0197 | 0.11971 | 239 R.SYELPDGQVITIGNER.F 256 |
CDNA FLJ41329 fis, clone BRAMY2047676 | Q6ZWC4 | none | 1.9 | 3.05 | 0.00548 | 1546.8087 | 0.08170 | 107 R.ALNLGAATVLRRHR.A 122 |
SLC22A11 | Q9NSA0 | Isoform 1 of Solute carrier family 22 member 11 | 1.9 | 2.63 | 0.00783 | 2210.0904 | 0.46292 | 301 R.INGHKEAKNLTIEVLMSSVK.E 322 |
RPS27A | P62979 | UBC;UBB ubiquitin and ribosomal protein S27a precursor | 1.8 | 3.39 | 0.01933 | 1788.1465 | 0.15637 | 11 K.TITLEVEPSDTIENVK.A 28 |
ARL6IP5 | O75915 | PRA1 family protein 3 | 1.8 | 2.85 | 0.00053 | 1312.8985 | 0.35814 | 9 R.AWDDFFPGSDR.F 21 |
ACTB | P60709 | Actin, cytoplasmic 1 | 1.8 | 4.57 | 0.00029 | 1132.7391 | 0.08870 | 196 R.GYSFTTTAER.E 207 |
GPR179 | A8MWI1 | Probable G-protein coupled receptor 179 precursor | 1.7 | 4.26 | 0.00002 | 1150.9204 | 0.12430 | 2099 R.GSSEAAGSVETR.V 2112 |
HSPA9 | P38646 | heat shock 70kDa protein 9, mitochondrial precursor | 1.7 | 4.66 | 0.04236 | 1450.7788 | 0.13564 | 85 R.TTPSVVAFTADGER.L 100 |
FAM62A | Q9BSJ8 | Isoform 1 of Protein FAM62A | 1.6 | 2.68 | 0.02300 | 1402.3521 | 0.45065 | 106 R.QLLDDEEQLTAK.T 119 |
ELMO2 | Q7Z5G9 | ELMO2 protein | 1.6 | 3.18 | 0.01234 | 1484.9090 | 0.09548 | 1 MERTQSSNMETR.L 13 |
CCDC73 | Q6ZRK6 | Isoform 1 of Coiled-coil domain-containing protein 73 | 1.6 | 2.39 | 0.01896 | 1583.8480 | 0.08212 | 84 K.EAMAVFKKQLQMK.M 98 |
PRPH | P41219 | Isoform 1 of Peripherin | 0.60 | 2.88 | 0.01008 | 1309.9330 | 0.17343 | 398 K.LLEGEESR.I 407 |
ATP5A1 | P25705 | ATP synthase subunit alpha, mitochondrial precursor | 0.60 | 2.54 | 0.00232 | 1316.9060 | 0.22248 | 218 K.TSIAIDTIINQK.R 231 |
CANX | B4DGP8 | Calnexin precursor | 0.37 | 2.70 | 0.00003 | 1488.7949 | 0.25015 | 480 R.IVDDWANDGWGLK.K 494 |
AKNA | Q7Z591 | Isoform 1 of AT-hook-containing transcription factor | 0.33 | 3.75 | 0.00007 | 1236.9160 | 0.16416 | 330 R.PLPRQGATLAGR.S 342 |
ITGB2(CD18) | P05107 | Integrin beta-2 precursor | 0.24 | 2.54 | 0.00017 | 1090.0657 | 0.37385 | 155 R.ALNEITESGR.I 166 |
a: average expression ratio;
b: cross-correlation score.
Among the thirty proteins identified, two proteins, CD18 and AKNA, were found to have a more than three-fold difference between active VKH patients and normal controls. The different expression of CD18 in both samples was shown in magnified intensity graphs (
Antibodies were used at a dilution of 1∶1000 for the anti-human CD18 monoclonal antibody and anti-human AKNA monoclonal antibody. Proteins were detected using the Phototope-HRP Western blot detection system(A). The immunoreactive band intensities were quantitated and were presented as intensity volumes (vol%). The results showed that both CD18 and AKNA were significantly down-regulated in VKH patients as compared to normal controls (B). VKH: VKH patients, NC: normal controls.
In this study, label-free relative quantitative proteomics was employed to screen differently expressed membrane proteins in CD4+ T cells from active VKH patients and normal controls. Thirty proteins were identified to be at least 1.5 fold differently expressed between active VKH patients and normal individuals. A significantly decreased expression of CD18 and AKNA was further confirmed using the Western blot technique. The results presented here may provide new clues for the study on molecules involved in the pathogenesis of VKH syndrome.
CD4+ T cells have been shown to be crucial in the pathogenesis of this syndrome
To ensure the accuracy and stability of the label-free quantitative analysis, several procedures were performed. For the purpose of minimizing the effect of individual differences on the experimental results, an equal amount of membrane proteins of CD4+ T cells from five active VKH patients and five normal controls were pooled respectively and subjected to the label-free quantitative proteomics. For label-free proteomics, strategies using three or four technical replicates have been reported
Our label-free proteomic results showed that thirty proteins were different in expression for more than 1.5 fold between active VKH patients and normal controls,although these results need to be confirmed by other techniques. By searching for the functions of these proteins according to annotations in the Swiss-prot database at
By analyzing the data acquired using the label-free method, it was interesting to note that a number of proteins identified in this study, including vimentin and AKNA transcription factor, were non-membrane components. A similar result has also been observed in proteomic studies. These non-membrane molecules could be explained as being a component associated with the membrane or with membrane fractions
False positive data always exist in proteomic studies. Therefore, the proteomic results usually need to be confirmed using other techniques. As protein with a big different expression ratio is readily confirmed by another technique, we chose two proteins with a three-fold different expression between VKH patients and normal controls, CD18 and AKNA, as candidates in the Westernblot identification. As expected, the Western blot result revealed a five-fold lower expression of both proteins in active VKH patients as compared with normal controls. It is worthwhile to point out that both CD18 and AKNA were identified using the one peptide based identification, as already reported previously
CD18, also known as integrin beta 2 or ITGB2, is one of the members of the integrin family, which is involved in cell adhesion, neutrophil chemotaxis, and cell-surface mediated signaling. The expression of CD18 can be down-regulated by introducing an insertion mutation in the murine CD18 gene. Due to this mutation, a chronic inflammatory skin disease clinically resembling human psoriasis develops in PL/J mice
ANKA, also known as AHCTF1, is reported to regulate the expression of CD40 and CD40L
In summary, label-free quantitative proteomics showed abnormal expression of thirty proteins for more than 1.5 fold in active VKH patients. A significantly decreased expression of CD18 and AKNA was confirmed using the Western blot technique. Both proteins may be involved in the pathogenesis of VKH syndrome. However, our study did not elucidate the mechanism by which both proteins exert their function. Further studies about the effect of both proteins on cytokine expression, apoptosis and chemotaxis of CD4+ T cells will contribute to our understanding about the mechanisms in the pathogenesis of VKH syndrome.
A representative binary comparison map of duplicate runs of samples from VKH patients. The horizontal (x) axis represents the distribution of peak intensities of the first run, and the vertical (y) axis represents that of the second run. The average intensity correlation coefficient (CC) between the two runs was 0.92. The expected distribution of the duplicate runs showed no obvious change.
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A representative base peak ion chromatogram from LC-MS/MS analysis of a total membrane protein extract from the VKH group digested with trypsin.
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The MS/MS spectra for single-peptide-based identifications. The detected b and y ions used in the protein identification were labeled in the MS/MS spectra. The unlabeled peaks are a, c, x or z ions generated in mass spectrometer.
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All the identified peptides and their sequences of each protein in both samples.
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C.V.analysis of all the identified proteins based on at least two peptides.The average CV of the fold changes was 23% for proteins with two or more peptides.
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Biological Process of the identified proteins.
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The molecular function of the identified proteins.
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