Proteomic Analysis of the Serum of Patients with Stable Vitiligo and Progressive Vitiligo

Abstract

To the Editor: Vitiligo is not a fatal disease; however, it can have strong social and psychological impacts on the patients. Genetic susceptibility, autoimmunity, neural dysregulation, melanin self-destruction, and oxidative stress may be involved in the pathogenesis of vitiligo. Based on its clinical development, vitiligo can be divided into the progressive vitiligo (PV) or the stable vitiligo (SV). To date, at least 50 susceptible genes had been found in vitiligo.[1] However, only a few genes presented a clear association with the high inherent risk of vitiligo and the protein levels of these genes were scarce. Recently, combination methods had been introduced in order to obtain better results. Nevertheless, no validated tool could confirm on the stage of vitiligo. Proteomics is a useful tool for large-scale screening of disease-related proteins. It could provide a better understanding of the biological and molecular events for this disease. Isobaric tag for relative and absolute quantitation (iTRAQ) is a systematic protein quantitative analytical method. It has the highest flux, the smallest system error, and the most powerful function for diagnosis, treatment, and prognosis. In this study, we identified differentially expressed proteins using iTRAQ-based proteomic technology and constructed an interaction network for SV and PV with the aim to identify potential group proteins as markers to distinguish between SV and PV. All the patients and healthy controls were enrolled at the Dermatological Outpatient Clinic of the First Hospital of China Medical University from 2015 to 2016. Vitiligo was diagnosed clinically by experienced dermatologists. Staging of the disease was performed according to the Vitiligo Disease Activity Score (VIDA) in the Consensus of Vitiligo Diagnosis and Treatment (2014 edition) issued by the Dermatology Committee of Pigmentary Disease of the Chinese Association of Integrative Medicine. Patients with PV (VIDA score over 2 points) showed emergence of new skin lesions expansion of the original skin lesions or occurrence of the Koebner phenomenon within three months. Patients with SV were defined as those with stable lesions for at least one year. The sites and progression of the lesions and the extent of cutaneous involvement were documented. Clinical information of the patients with PV and SV are presented in Supplementary Tables 1 and 2. Ten serum samples from each group were pooled together for proteomic experiments. The remaining 20 serum samples were used in Western blotting experiments for verification of results. Supplementary Table 1 Characteristics of patients with PV Number Gender Age (years) Time of new-onset Site Koebner phenomenon Family history Patient 1 Male 17 Nearly 1 month e, f, k, l Yes No Patient 2 Male 29 Nearly 3 months a, b, c, e, f Yes Yes Patient 3 Female 33 Nearly 1 month a, b, c, e, f, g, j, k, l Yes No Patient 4 Female 24 Nearly 1 month e, f, k, l Yes No Patient 5 Female 19 Nearly 1 month a, b, c, f, k, l No Yes Patient 6 Male 41 Nearly 4 months a, b, c, g, k, l Yes No Patient 7 Female 38 Nearly 2 months a, b, d, e, f, k, l Yes No Patient 8 Male 29 Nearly 1 month g, i, j No No Patient 9 Female 33 Nearly 3 months a, b, c, d, e, f, g, k, l Yes No Patient 10 Male 26 Nearly 6 months e, f, k, l Yes No The VIDA integral: Nearly 6 weeks appear new lesions or lesions expand (+4), nearly 3 months appear new lesions or lesions expand (+3), nearly 6 months appear new lesions or lesions expand (+2); nearly 1 year appear new lesions or lesions expand (+1). Score >1 points is progression, >4 points for the rapid progression. All patients have no autoimmune disease. Vitiligo in the progressive stage included 12 cases of sporadic vitiligo (40.0%), 10 cases of acrofacial (33.3%), and 8 cases of universal (26.7%), they are 5 males and 5 females. a: Head (3%); b: Face (3%); c: Neck (3%); d: Upperarm (7%); e: Forearm (6%); f: Hands (5%); g: Trunk (26%); h: Perineum (1%); i: Hip (5%); j: Thigh (21%); k: Shin (13%); l: Feet (7%). VIDA: Vitiligo Disease Activity Score; PV: Progressive vitiligo. Supplementary Table 2 Characteristics of patients with SV Number Gender Age Time of stable phase (years) Site Koebner phenomenon Family history Patient 11 Male 27 >10 e, f, k, l No Yes Patient 12 Female 38 >10 a, b, c, e, f, g, i, j, k, l No No Patient 13 Female 43 >20 a, b, c, e, f, l No No Patient 14 Female 20 >4 a, b, c, e, f No No Patient 15 Female 16 >5 a, b, c, f, k, l No No Patient 16 Male 33 >10 a, b, c, e, f, k, l No No Patient 17 Male 47 >10 a, b, c, d, e, f, g, k, l No No Patient 18 Female 34 >20 g, i, j No Yes Patient 19 Female 26 >10 g, i, j, k, l No No Patient 20 Male 29 >10 a, b, c, e, f, k, l No No The evaluation of the criterion in SV is set by disease activity score (VIDA). Depigmentation phase was stable for >1 year (0 score), at the same time, there is spontaneous pigment regeneration (−1). All patients have no autoimmune disease. Vitiligo in the stable stage included 14 cases of sporadic vitiligo (46.7%), 8 cases of acrofacial (26.7%), 8 cases of universal (26.7%), they are 4 males and 6 females. a: Head (3%); b: Face (3%); c: Neck (3%); d: Upperarm (7%); e: Forearm (6%); f: Hands (5%); g: Trunk (26%); h: Perineum (1%); i: Hip (5%); j: Thigh (21%); k: Shin (13%); l: Feet (7%). VIDA: Vitiligo disease activity score; SV: Stable vitiligo. Pooled serum samples from each group were prepared before proteomic analysis. The ProteoPrep Blue Albumin and IgG Depletion kit (Sigma-Aldrich, Co., St. Louis, MO, USA) were used to remove albumin, and IgG protein concentration was estimated using the BCA Protein Assay Kit (Thermo Scientific, Rockford, California, USA). Total proteins of each group were analyzed by iTRAQ (AB Sciex, Framingham, Massachusetts, USA), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Protein samples (100 μg) were reduced, alkylated, and subjected to tryptic hydrolysis. iTRAQ labeling was performed using iTRAQ Reagents manufacturer's protocol. Each sample was labeled with the respective iTRAQ tags. All the labeled peptides were merged and evaporated to dryness in a vacuum centrifuge. The iTRAQ labelled samples were firstly diluted to 100 μl with H2O buffer (NH3•H2O, pH=10) before high performance liquid chromatography on a Dionex Ultimate 3000 system (Dionex, Sunnyvale, CA, USA) at 25°C on a Gemini NX 3u C18 110A; 150.0 mm × 2.0 mm Phenomenex column, and Gemini 3u C6 Phenyl 110A; 100.0 mm × 2.0 mm column (all from Phenomenex, Torrance, CA, USA). The flow rate used for reversed-phase column separation was 0.2 ml/min with H2O (mobile Phase A) and 80% ACN (mobile Phase B). A solvent gradient system was used: 0–15 min, 5–10% B; 15–48 min, 15–25% B; 48–60 min, 25–37% B; 60–65 min, 37–95% B; and 65–70 min, 95% B. The elution was monitored by absorbance at 214/280 nm, and fractions were collected every 50 s. In total, 10 fractions were combined and dried. Peptides were separated by a linear gradient according to the manufacture's instruction. MS analysis was performed on a Q Exactive system (Thermo Fisher Scientific, California, USA) in information-dependent mode. MS spectra were acquired across the mass range of 350–1800 m/z in high-resolution mode (>35,000); a maximum of 20 precursors per cycle were chosen for fragmentation from each MS spectrum with a 120-ms minimum accumulation time for each precursor and dynamic exclusion for 10s. The tandem mass spectra were recorded in high-sensitivity mode (resolution >175,000) with rolling collision energy and iTRAQ reagent collision energy adjustments. Proteins extracted from the serum samples of 20 patients with SV, 20 patients with PV, and 20 healthy controls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11–15% acrylamide). After transferring the blots onto PVDF membranes and blocking overnight (5% skim milk and 0.05% Tween-20 in PBS), the primary antibody was added for 1 h, followed by PBS washing, addition of a secondary HRP-conjugated antibody, and development with a chemiluminescence detection system (ECL, Pierce, USA). Anti-plasma serine protease inhibitor (SERPINA5), hepatocyte growth factor activator (HGFAC), anti-leucine zipper protein 4 (LUZP4), and anti-phosphoinositide phospholipase C (PLCH2) were used (Abcam, USA). The interaction network of differentially expressed proteins was constructed by the Cytoscape (National Institute of General Medical Sciences, USA), which is an open source software platform for visualizing molecular interaction networks and biological pathways and integrating these networks with annotations. The differentially expressed proteins involved in biological processes, molecular functions, cell components, and pathway enrichment were evaluated using the Cytoscape platform based on Gene Ontology (GO) terms. The corresponding P value analysis was simultaneously obtained using ReactomeFIViz (National Institute of General Medical Sciences). The peptide data were analyzed with Protein Pilot Software 5.0 using the Paragon protein database search algorithm (AB Sciex, Framingham, Massachusetts, USA). The resulting MS/MS spectra were searched against the International Protein Index human sequence database (version 3.83). The parameters for the analysis were set as follows: Cys alkylation with methyl methanethiosulfonate, digestion with trypsin, and allowance of up to one missed trypsin cleavage. The false discovery rate (FDR) analysis was also performed using the integrated tools (FDR ≤0.01). All the data were analyzed using Statistical Package for the Social Sciences Software (SPSS Inc., Chicago, IL, USA) version 16.0. The significant difference was analyzed by one-way analysis of variance (ANOVA). The gray value ratio of each band compared between the groups (SV or PV compared to control) was used to calculate the significant difference by least significant difference as a post hoc test. A value of P < 0.05 was considered statistically significant. In total, 171 differentially expressed proteins were identified through iTRAQ. Compared with the control group, there were 80 (42 upregulated and 38 downregulated) and 89 (56 upregulated and 33 downregulated) differentially expressed proteins in the SV and PV groups, respectively [Supplementary Tables 3 and 4]. Among these differentially expressed proteins, 39 showed similar changes in both the SV and PV groups. Among them, the differentially expressed upregulated and downregulated proteins were 19 and 20. When the progressive stage was compared with the stable stage of vitiligo, 71 (23 upregulated and 48 downregulated) proteins were found to be differentially expressed [Supplementary Table 5]. Supplementary Table 3 Identification of differentially expressed proteins of SV samples versus control samples Accession number Protein name Gene name Mw pI Coverage (%) Peptides (95%) Unused protscore SV ratio B9DI82 Phosphoinositide phospholipase C (fragment) PLCH2 134,778.10 7.64 3.15 1 1.8 5.73 P05154 Plasma serine protease inhibitor SERPINA5 45,674.14 9.30 21.92 7 11.73 10.07 A0A0B4J2B7 Protein IGHV3-30 (fragment) IGHV3-30 12,946.63 9.10 84.62 39 4 5.29 P23083 Ig heavy chain V-I region V35 N/A 13,008.57 9.59 73.50 28 6.08 3.27 P06331 Ig heavy chain V-II region ARH-77 N/A 16,228.27 8.45 26.71 29 2 3.56 A0A0C4DH31 Protein IGHV1-18 (fragment) IGHV1-18 12,820.29 8.98 55.56 20 6.01 2.82 P01876 Ig alpha-1 chain C region IGHA1 37,654.23 6.08 79.04 191 35.61 4.23 A0A075B6K4 HCG2043238 (fragment) IGLV3-10 12,628.89 4.72 59.83 17 10.2 2.37 A0A0B4J2H0 Protein IGHV1-69-2 (fragment) IGHV1-69-2 12,660.18 8.64 59.83 33 4 1.80 Q15485 Ficolin-2 FCN2 34,000.76 6.31 24.60 9 8.1 2.59 A0A075B6K9 Ig lambda-2 chain C regions (fragment) IGLC2 11,236.38 6.91 99.06 84 20.51 3.40 A0A0B4J1V0 Protein IGHV3-15 (fragment) IGHV3-15 12,925.64 8.84 78.15 20 8.1 1.98 A0A0C4DH67 Protein IGKV1-8 (fragment) IGKV1-8 12,537.18 9.21 58.26 18 4.27 1.83 A0A075B6N7 Ig alpha-2 chain C region (fragment) IGHA2 36,590.95 5.86 67.35 72 10.04 2.33 D6RF35 Vitamin D-binding protein GC 53,020.02 5.38 84.45 81 4.01 1.81 P06319 Ig lambda chain V-VI region EB4 N/A 14,146.57 4.85 39.69 10 4.87 1.69 P81605 Dermcidin DCD 11,283.74 6.09 20.91 1 2.04 1.51 Q08380 Galectin-3-binding protein LGALS3BP 65,330.25 5.13 29.06 10 14.59 1.88 Q04756 Hepatocyte growth factor activator HGFAC 70,681.11 6.99 20.00 14 16.16 1.82 P03951 Coagulation factor XI F11 70,108.40 8.47 17.76 8 8.34 1.75 P01709 Ig lambda chain V-II region MGC N/A 11,557.43 5.12 42.34 2 2 1.92 P06316 Ig lambda chain V-I region BL2 N/A 13,564.07 7.64 62.31 12 4.11 1.85 P35908 Keratin, type II cytoskeletal 2 epidermal KRT2 65,432.11 8.07 24.10 13 17.64 1.65 A0A0B4J231 Immunoglobulin lambda-like polypeptide 5 IGLL5 23,150.06 9.08 55.35 63 4.32 2.09 A0A0C4DH41 Protein IGHV4-61 (fragment) IGHV4-61 13,065.75 9.36 55.93 35 13.04 1.57 A0A0G2JN06 Ig gamma-2 chain C region (fragment) IGHG2 35,900.23 7.66 79.75 142 14.68 1.51 A0A0C4DH38 Protein IGHV5-51 (fragment) IGHV5-51 12,674.41 8.45 70.09 25 8.07 1.57 Q71DI3 Histone H3.2 HIST2H3A 15,387.84 11.27 34.56 3 3.9 1.74 A0A0B4J1U2 Protein IGLV7-43 (fragment) IGLV7-43 12,450.88 6.52 16.24 5 3.06 1.57 B4E1Z4 Uncharacterized protein N/A 140,941.20 6.82 54.58 104 101.59 1.57 P00734 Prothrombin F2 70,036.12 5.64 72.19 69 56.96 1.70 P02042 Hemoglobin subunit delta HBD 16,055.28 7.84 90.48 33 11.89 1.64 P01780 Ig heavy chain V-III region JON N/A 12,319.98 9.39 60.87 10 2 1.70 A0A087WYJ9 Ig mu chain C region IGHM 65,700.17 6.52 66.44 118 55.83 1.55 P02766 Transthyretin TTR 15,886.83 5.52 72.11 23 18.87 1.58 P01703 Ig lambda chain V-I region NEWM N/A 10,904.07 9.39 78.64 22 2.01 1.96 P04196 Histidine-rich glycoprotein HRG 59,577.63 7.09 46.67 24 24.58 1.59 P00747 Plasminogen PLG 90,568.09 7.04 62.35 94 74.79 1.67 P01031 Complement C5 C5 188,303.00 6.11 44.93 66 91.04 1.57 P14780 Matrix metalloproteinase-9 MMP9 78,457.32 5.69 12.31 6 10.76 1.58 A0A0C4DH68 Protein IGKV2-24 (Fragment) IGKV2-24 13,078.87 8.74 55.83 20 4.71 1.63 P69905 Hemoglobin subunit alpha HBA1 15,257.36 8.72 60.56 24 9.73 1.54 Q9P127 Leucine zipper protein 4 LUZP4 35,936.42 9.47 3.83 2 2 −4.09 P35527 Keratin, type I cytoskeletal 9 KRT9 62,063.62 5.14 19.90 13 14.49 −4.10 P02763 Alpha-1-acid glycoprotein 1 ORM1 23,511.27 4.93 53.73 57 22.45 −2.98 P69891 Hemoglobin subunit gamma-1 HBG1 16,140.27 6.64 45.58 14 4.7 −3.12 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 101,387.90 6.31 50.27 87 53.78 −1.84 P0C0L4 Complement C4-A C4A 192,783.20 6.66 71.90 219 181.76 −1.66 P27169 Serum paraoxonase/arylesterase 1 PON1 39,730.81 5.08 62.82 40 23.94 −2.23 P02748 Complement component C9 C9 63,172.73 5.43 49.37 32 35.29 −2.69 O75636 Ficolin-3 FCN3 32,902.61 6.20 36.12 15 13.22 −3.20 P35858 Insulin-like growth factor-binding protein complex acid labile subunit IGFALS 66,034.20 6.33 30.08 13 23.34 −2.03 P02750 Leucine-rich alpha-2-glycoprotein LRG1 38,177.44 6.45 37.46 15 17.79 −2.02 P07996 Thrombospondin-1 THBS1 129,381.40 4.71 30.94 37 50.97 −2.17 P04259 Keratin, type II cytoskeletal 6B KRT6B 60,066.25 8.09 23.05 10 8.03 −2.16 P19652 Alpha-1-acid glycoprotein 2 ORM2 23,602.35 5.03 54.23 33 9.59 −2.20 P0DJI8 Serum amyloid A-1 protein SAA1 13,531.86 6.28 63.11 6 2.91 −2.19 P20742 Pregnancy zone protein PZP 16,3861.00 5.97 37.52 119 31.65 −2.34 P02741 C-reactive protein CRP 25,038.26 5.45 23.66 3 3.3 −2.07 P18428 Lipopolysaccharide-binding protein LBP 53,382.97 6.23 18.09 9 12.92 −2.20 A0A0A6YYG9 Protein ARPC4-TTLL3 ARPC4-TTLL3 71,718.39 5.59 6.40 2 2 −1.57 Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 103,356.20 6.51 65.59 110 85.05 −1.77 Q13201 Multimerin-1 MMRN1 138,108.60 8.15 8.63 5 5.48 −1.93 P01011 Alpha-1-antichymotrypsin SERPINA3 47,650.31 5.33 66.19 86 43.42 −1.63 P04264 Keratin, type II cytoskeletal 1 KRT1 66,037.95 8.15 29.35 21 26.61 −1.82 A0A075B6K3 Protein IGLV2-11 (fragment) IGLV2-11 12,643.88 6.69 51.26 5 4 −1.84 P23142 Fibulin-1 FBLN1 77,213.47 5.07 29.16 16 21.44 −1.89 Q16610 Extracellular matrix protein 1 ECM1 60,673.46 6.25 34.44 12 19.14 −1.68 P04275 von Willebrand factor VWF 309,262.00 5.29 16.74 27 50.73 −1.57 P02647 Apolipoprotein A-I APOA1 30,777.44 5.56 88.39 228 77.72 −1.53 E7ES19 Thrombospondin-4 THBS4 96,004.91 4.39 10.46 4 3.04 −1.62 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 106,462.20 6.40 49.79 90 49.73 −1.58 P06681 Complement C2 C2 83,266.95 7.23 41.09 30 10.01 −1.73 P01009 Alpha-1-antitrypsin SERPINA1 46,735.98 5.37 88.28 240 91.91 −1.49 P25311 Zinc-alpha-2-glycoprotein AZGP1 34,258.30 5.71 64.09 35 37.02 −1.73 V9GYM3 Apolipoprotein A-II APOA2 14,914.10 8.43 63.16 52 28.49 −1.62 A0A087WXI2 IgGFc-binding protein FCGBP 44,5207.00 5.16 7.45 10 18.84 −1.63 P07339 Cathepsin D CTSD 44,551.72 6.10 11.89 2 2.02 −1.61 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 99,848.02 5.49 38.20 24 22.37 −1.52 P55058 Phospholipid transfer protein PLTP 54,738.80 6.53 31.03 10 15.38 −1.61 Mw: Molecular weight; PV: Progressive vitiligo; SV: Stable vitiligo; N/A: Not applicable; pI: Isoelectric point. Supplementary Table 4 Identification of differentially expressed proteins of PV samples versus control samples Accession number Protein name Gene name Mw pI Coverage (%) Peptides (95%) Unused protscore PV ratio A0A024R6I7 Alpha-1-antitrypsin SERPINA1 46,707.93 5.37 87.80 230 2 39.25 P05154 Plasma serine protease inhibitor SERPINA5 45,674.14 9.30 21.92 7 11.73 10.53 Q04756 Hepatocyte growth factor activator HGFAC 70,681.11 6.99 20.00 14 16.16 2.55 P00739 Haptoglobin-related protein HPR 39,029.11 6.63 81.03 100 14.38 2.41 P08779 Keratin, type I cytoskeletal 16 KRT16 51,267.25 4.98 27.27 9 9.89 2.91 P69905 Hemoglobin subunit alpha HBA1 15,257.36 8.72 60.56 24 9.73 2.73 P03951 Coagulation factor XI F11 70,108.40 8.47 17.76 8 8.34 2.08 Q15485 Ficolin-2 FCN2 34,000.76 6.31 24.60 9 8.1 2.89 A0A0B4J2H0 Protein IGHV1-69-2 (fragment) IGHV1-69-2 12,660.18 8.64 59.83 33 4 1.70 P01876 Ig alpha-1 chain C region IGHA1 37,654.23 6.08 79.04 191 35.61 4.58 P80108 Phosphatidylinositol-glycan- specific phospholipase D GPLD1 92,335.35 5.91 28.45 17 30.27 1.84 P04114 Apolipoprotein B-100 APOB 515,598.30 6.58 66.10 550 468.39 1.89 K7ER74 Protein APOC4-APOC2 APOC4-APOC2 20,049.07 6.36 51.69 21 16 2.17 P06331 Ig heavy chain V-II region ARH-77 N/A 16,228.27 8.45 26.71 29 2 2.45 A0A0B4J231 Immunoglobulin lambda-like polypeptide 5 IGLL5 23,150.06 9.08 55.35 63 4.32 2.55 B0YIW2 Apolipoprotein C-III APOC3 12,815.43 7.90 47.01 36 12.61 2.47 P02042 Hemoglobin subunit delta HBD 16,055.28 7.84 90.48 33 11.89 2.10 P23083 Ig heavy chain V-I region V35 N/A 13,008.57 9.59 73.50 28 6.08 1.94 P0C0L5 Complement C4-B C4B 192,749.20 6.89 71.56 220 8.82 2.77 P55056 Apolipoprotein C-IV APOC4 14,552.90 9.19 49.61 6 5.38 2.10 P08697 Alpha-2-antiplasmin SERPINF2 54,565.11 5.87 43.79 29 19.89 1.58 P06396 Gelsolin GSN 85,696.47 5.90 53.07 53 47.75 2.02 A0A075B6N7 Ig alpha-2 chain C region (fragment) IGHA2 36,590.95 5.86 67.35 72 10.04 2.24 P00734 Prothrombin F2 70,036.12 5.64 72.19 69 56.96 1.81 P68871 Hemoglobin subunit beta HBB 15,998.21 6.74 82.31 43 24.33 2.09 P10909 Clusterin CLU 52,494.01 5.89 51.22 63 34.68 1.65 P04196 Histidine-rich glycoprotein HRG 59,577.63 7.09 46.67 24 24.58 2.17 B4E1Z4 Uncharacterized protein N/A 140,941.20 6.82 54.58 104 101.59 2.14 P19652 Alpha-1-acid glycoprotein 2 ORM2 23,602.35 5.03 54.23 33 9.59 1.77 F5H7G1 Complement component C8 beta chain C8B 61,230.52 7.86 30.61 9 18.49 2.48 P00742 Coagulation factor X F10 54,731.14 5.68 37.50 13 23.06 1.73 A0A0C4DH41 Protein IGHV4-61 (fragment) IGHV4-61 13,065.75 9.36 55.93 35 13.04 1.60 P08519 Apolipoprotein (a) LPA 501,314.20 5.58 39.78 20 29.93 1.56 A0A075B6K9 Ig lambda-2 chain C regions (fragment) IGLC2 11,236.38 6.91 99.06 84 20.51 2.01 P08603 Complement factor H CFH 139,095.00 6.21 59.38 138 120.45 1.76 P04259 Keratin, type II cytoskeletal 6B KRT6B 60,066.25 8.09 23.05 10 8.03 1.55 Q12805 EGF-containing fibulin-like extracellular matrix protein 1 EFEMP1 54,640.10 4.95 14.81 5 8.01 1.69 E9PHK0 Tetranectin CLEC3B 17,793.93 4.96 66.25 15 10.07 1.59 P08493 Matrix Gla protein MGP 12,353.06 9.71 14.56 1 2 1.50 P43652 Afamin AFM 69,068.41 5.64 58.93 46 52.35 1.99 O00391 Sulfhydryl oxidase 1 QSOX1 82,576.74 9.13 14.59 4 7.52 1.77 P20851 C4b-binding protein beta chain C4BPB 28,357.18 5.05 38.89 7 9.3 1.68 P01031 Complement C5 C5 188,303.00 6.11 44.93 66 91.04 1.94 A0A087WTM7 Apolipoprotein B-100 APOB 489,827.10 6.69 67.61 536 2 1.61 P01011 Alpha-1-antichymotrypsin SERPINA3 47,650.31 5.33 66.19 86 43.42 1.53 A0A075B6K4 HCG2043238 (fragment) IGLV3-10 12,628.89 4.72 59.83 17 10.2 1.58 P14780 Matrix metalloproteinase-9 MMP9 78,457.32 5.69 12.31 6 10.76 1.66 P05546 Heparin cofactor 2 SERPIND1 57,069.95 6.41 58.72 52 41.5 1.75 P02765 Alpha-2-HS-glycoprotein AHSG 39,324.24 5.43 64.31 72 35.09 1.73 P01019 Angiotensinogen AGT 53,153.57 5.87 41.24 32 21.65 1.67 Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 103,356.20 6.51 65.59 110 85.05 1.73 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 101,387.90 6.31 50.27 87 53.78 1.62 A0A0C4DH68 Protein IGKV2-24 (fragment) IGKV2-24 13,078.87 8.74 55.83 20 4.71 1.59 P02647 Apolipoprotein A-I APOA1 30,777.44 5.56 88.39 228 77.72 1.56 P05160 Coagulation factor XIII B chain F13B 75,509.92 6.01 50.08 31 42 1.53 P06316 Ig lambda chain V-I region BL2 N/A 13,564.07 7.64 62.31 12 4.11 1.66 Q14019 Coactosin-like protein COTL1 15,944.79 5.54 11.27 1 2 −14.79 A0A087WYJ9 Ig mu chain C region IGHM 65,700.17 6.52 66.44 118 55.83 −9.37 P01591 Immunoglobulin J chain JCHAIN 18,098.40 5.12 70.44 15 9.66 −5.02 Q92820 Gamma-glutamyl hydrolase GGH 35,963.95 6.66 29.25 4 6.3 −2.55 O43866 CD5 antigen-like CD5L 38,087.45 5.28 44.96 18 21.83 −2.96 Q9P127 Leucine zipper protein 4 LUZP4 35,936.42 9.47 3.83 2 2 −3.96 P04278 Sex hormone-binding globulin SHBG 43,778.68 6.22 34.33 10 13.7 −2.67 A0A0C4DH43 Uncharacterized protein (fragment) N/A 13,312.31 8.50 35.29 2 2.07 −3.28 A0A0G2JPD4 Ig gamma-4 chain C region (fragment) IGHG4 35,940.14 7.18 73.09 175 6.07 −2.35 P02750 Leucine-rich alpha-2-glycoprotein LRG1 38,177.44 6.45 37.46 15 17.79 −2.36 A0A0B4J1X5 Protein IGHV3-74 (fragment) IGHV3-74 12,839.47 8.91 83.76 104 3.38 −4.07 P05109 Protein S100-A8 S100A8 10,834.38 6.51 39.78 6 8.75 −2.39 P07996 Thrombospondin-1 THBS1 129,381.40 4.71 30.94 37 50.97 −2.06 P00738 Haptoglobin HP 45,204.78 6.13 88.18 235 70.99 −1.85 P06702 Protein S100-A9 S100A9 13,241.85 5.71 49.12 4 7.05 −2.00 A0A0C4DH67 Protein IGKV1-8 (fragment) IGKV1-8 12,537.18 9.21 58.26 18 4.27 −1.84 P40197 Platelet glycoprotein V GP5 60,958.46 9.73 12.86 4 4.72 −1.92 Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 99,848.02 5.49 38.20 24 22.37 −2.05 Q8IVF4 Dynein heavy chain 10, axonemal DNAH10 514,834.70 5.64 6.15 3 2.03 −2.03 A0A087WUS7 Ig delta chain C region IGHD 42,352.35 8.38 42.71 16 11.8 −2.75 P07339 Cathepsin D CTSD 44,551.72 6.10 11.89 2 2.02 −2.02 O75636 Ficolin-3 FCN3 32,902.61 6.20 36.12 15 13.22 −1.64 P35527 Keratin, type I cytoskeletal 9 KRT9 62,063.62 5.14 19.90 13 14.49 −1.71 P02776 Platelet factor 4 PF4 10,844.83 8.93 29.70 2 4 −1.80 P02748 Complement component C9 C9 63,172.73 5.43 49.37 32 35.29 −1.50 P69891 Hemoglobin subunit gamma-1 HBG1 16,140.27 6.64 45.58 14 4.7 −1.52 P83593 Ig kappa chain V-IV region STH (fragment) N/A 12,060.29 7.94 71.56 16 2.01 −1.77 P0DJI8 Serum amyloid A-1 protein SAA1 13,531.86 6.28 63.11 6 2.91 −1.70 P18428 Lipopolysaccharide-binding protein LBP 53,382.97 6.23 18.09 9 12.92 −1.51 P22792 Carboxypeptidase N subunit 2 CPN2 60,555.93 5.63 26.61 15 14.3 −1.70 P20742 Pregnancy zone protein PZP 163,861.00 5.97 37.52 119 31.65 −1.53 P01770 Ig heavy chain V-III region NIE N/A 12,898.45 9.75 54.62 24 6.02 −1.69 P06313 Ig kappa chain V-IV region JI N/A 14,632.35 6.15 71.43 51 13.34 −1.60 Mw: Molecular weight; PV: Progressive vitiligo; N/A: Not applicable; pI: Isoelectric point. Supplementary Table 5 Differentially expressed proteins occurring in PV compared with SV Accession number Protein name Gene name Mw Coverage (%) Peptides (95%) Unused protscore PV/SV ratio A0A087WYJ9 Ig mu chain C region IGHM 65,700.17 66.44 118 55.83 12.47 Q14019 Coactosin-like protein COTL1 15,944.79 11.27 1 2 19.41 A0A0B4J1X5 Protein IGHV3-74 (fragment) IGHV3-74 12,839.47 83.76 104 3.38 5.97 P01591 Immunoglobulin J chain JCHAIN 18,098.40 70.44 15 9.66 4.83 A0A087WUS7 Ig delta chain C region IGHD 42,352.35 42.71 16 11.8 4.66 A0A0C4DH67 Protein IGKV1-8 (fragment) IGKV1-8 12,537.18 58.26 18 4.27 2.78 O43866 CD5 antigen-like CD5L 38,087.45 44.96 18 21.83 2.65 P04278 Sex hormone-binding globulin SHBG 43,778.68 34.33 10 13.7 2.23 A0A0C4DH31 Protein IGHV1-18 (fragment) IGHV1-18 12,820.29 55.56 20 6.01 2.75 P01770 Ig heavy chain V-III region NIE IGHA1 12,898.45 54.62 24 6.02 2.09 D6RF35 Vitamin D-binding protein GC 53,020.02 84.45 81 4.01 1.66 O43866 CD5 antigen-like CD5L 38,087.45 44.96 18 21.83 2.65 P04278 Sex hormone-binding globulin SHBG 43,778.68 34.33 10 13.7 2.23 A0A0C4DH43 Uncharacterized protein (fragment) N/A 13,312.31 35.29 2 2.07 3.70 A0A087WUS7 Ig delta chain C region IGHD 42,352.35 42.71 16 11.8 4.66 P05109 Protein S100-A8 S100A8 10,834.38 39.78 6 8.75 1.91 P01709 Ig lambda chain V-II region MGC N/A 11,557.43 42.34 2 2 1.85 P23083 Ig heavy chain V-I region V35 N/A 13,008.57 73.50 28 6.08 1.58 P01780 Ig heavy chain V-III region JON N/A 12,319.98 60.87 10 2 1.67 A0A0C4DH31 Protein IGHV1-18 (fragment) IGHV1-18 12,820 55.56 45 6.01 2.75 P06702 Protein S100-A9 S100A9 13,241.85 49.12 4 7.05 1.54 P60709 Actin, cytoplasmic 1 ACTB 41,737 6 31 35.33 1.63 A0A075B6K9 Ig lambda-2 chain C regions (fragment) IGLC2 11,236.38 99.06 84 20.51 1.91 A0A024R6I7 Alpha-1-antitrypsin SERPINA1 46,707.93 87.80 230 2 −51.52 P0C0L5 Complement C4-B C4B 19,2749.20 71.56 220 8.82 −2.86 P02763 Alpha-1-acid glycoprotein 1 ORM1 23,511.27 53.73 57 22.45 −3.94 P0C0L4 Complement C4-A C4A 192,783.20 71.90 219 181.76 −1.82 P19652 Alpha-1-acid glycoprotein 2 ORM2 23,602.35 54.23 33 9.59 −3.91 P33908 Mannosyl-oligosaccharide 1,2-alpha-mannosidase IA MAN1A1 72,969 13.17 4 6.27 −3.53 P04259 Keratin, type II cytoskeletal 6B KRT6B 60,066.25 23.05 10 8.03 −2.99 P19827 Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 101,387.90 50.27 87 53.78 −2.58 P01011 Alpha-1-antichymotrypsin SERPINA3 47,650.31 66.19 86 43.42 −2.13 P27169 Serum paraoxonase/arylesterase 1 PON1 39,730.81 62.82 40 23.94 −2.58 Q14624 Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 103,356.20 65.59 110 85.05 −3.49 F5H7G1 Complement component C8 beta chain C8B 61,230.52 30.61 9 18.49 −3.162 P23142 Fibulin-1 FBLN1 77,213.47 29.16 16 21.44 −1.74 P04275 von Willebrand factor VWF 309,262.00 16.74 27 50.73 −1.91 P08779 Keratin, type I cytoskeletal 16 KRT16 51,267.25 27.27 9 9.89 −3.22 P02741 C-reactive protein CRP 25,038.26 23.66 3 3.3 −2.88 P69891 Hemoglobin subunit gamma-1 HBG1 16,140.27 45.58 14 4.7 −1.87 P08519 Apolipoprotein (a) LPA 501,314.20 39.78 20 29.93 −1.82 P68871 Hemoglobin subunit beta HBB 15,998.21 82.31 43 24.33 −1.87 P35527 Keratin, type I cytoskeletal 9 KRT9 62,063.62 19.90 13 14.49 −2.81 P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 106,462.20 49.79 90 49.73 −1.91 P36980 Complement factor H-related protein 2 CFHR2 30,651 41.85 7 6 −1.91 Q16610 Extracellular matrix protein 1 ECM1 60,673.46 34.44 12 19.14 −2.07 P04264 Keratin, type II cytoskeletal 1 KRT1 66,037.95 29.35 21 26.61 −2.29 P02647 Apolipoprotein A-I APOA1 30,777.44 88.39 228 77.72 −2.94 P05546 Heparin cofactor 2 SERPIND1 57,069.95 58.72 52 41.5 −1.61 P06732 Creatine kinase M-type CKM 43,101 5.512 1 2 −1.50 P55056 Apolipoprotein C-IV APOC4 14,552.90 49.61 6 5.38 −1.80 Q9UHG3 Prenylcysteine oxidase 1 PCYOX1 56,640 15.45 5 8.78 −1.63 P01042 Kininogen-1 KNG1 71,957 55.12 106 54.98 −2.33 P06396 Gelsolin GSN 85,696.47 53.07 53 47.75 −1.51 P02748 Complement component C9 C9 63,172.73 49.37 32 35.29 −1.91 A0A0A0MR46 RNA-binding protein 44 RBM44 118,116 4.848 2 2 −1.54 P29622 Kallistatin SERPINA4 48,542 49.65 24 27.74 −1.74 P36955 Pigment epithelium-derived factor SERPINF1 46,312 57.89 17 26.25 −1.85 B0YIW2 Apolipoprotein C-III APOC3 12,815.43 47.01 36 12.61 −1.82 C9JF17 Apolipoprotein D (fragment) APOD 24,158 33.49 25 16.88 −3.25 P00450 Ceruloplasmin CP 12,2205 69.86 169 102.07 −1.89 P05155 Plasma protease C1 inhibitor SERPING1 55,154 55.6 49 36.17 −1.54 P06727 Apolipoprotein A-IV APOA4 45,399 78.79 71 53.32 −1.54 P20742 Pregnancy zone protein PZP 163,861.00 37.52 119 31.65 −1.57 P01009 Alpha-1-antitrypsin SERPINA1 46,735.98 88.28 240 91.91 −2.00 P05160 Coagulation factor XIII B chain F13B 75,509.92 50.08 31 42 −2.27 O00391 Sulfhydryl oxidase 1 QSOX1 82,576.74 14.59 4 7.52 −1.53 O75636 Ficolin-3 FCN3 32,902.61 36.12 15 13.22 −2.70 P02751 Fibronectin FN1 26,2625 55.28 186 151.63 −2.07 P02649 Apolipoprotein E APOE 36,154 50.08 31 42 −2.05 P00739 Haptoglobin-related protein HPR 39,029.11 81.03 100 14.38 −2.01 Mw: Molecular weight; PV: Progressive vitiligo; SV: Stable vitiligo; N/A: Not applicable. The differentially expressed proteins in the SV and PV groups were analyzed based on their GO clustering using the Cytoscape platform [Supplementary Figures 1 and 2]. The significance of the first 15 annotated functions was ranked according to the P values. The differently expressed proteins were categorized based on their molecular function, biological process, pathway enrichment, and cell component. Supplementary Figure 1 Categorization of differentially expressed proteins of progressive vitiligo compared to control. (a) The top 15 proteins with molecular function. (b) The top 15 proteins involved in biological processes. (c) The top 15 proteins in pathway enrichment. (d) The top 15 proteins of cell component. The log-transformed enrichment scores for molecular function, biological process, pathway enrichment, and cell component are indicated on the x axis. Click here for additional data file. Supplementary Figure 2 Categorization of differentially expressed proteins of stable vitiligo compared to control. (a) The top 15 proteins with molecular function. (b) The top 15 proteins involved in biological processes. (c) The top 15 proteins in pathway enrichment. (d) The top 15 proteins of cell component. The log-transformed enrichment scores for molecular function, biological process, pathway enrichment and cell component are indicated on the x axis. Click here for additional data file. In the SV group, molecular function of the differentially expressed proteins included immunoglobulin (Ig) receptor binding, antigen binding, phosphatidylcholine binding, serine-type endopeptidase inhibitor activity, endopeptidase inhibitor activity, Ig binding, protease binding, complement component C1q binding, serine-type endopeptidase activity, oxygen transporter activity, glycoprotein binding, heparin binding, proteoglycan binding, low-density lipoprotein particle binding, and laminin binding proteins, as compared to the controls. PV group expressed the above proteins similar to those in the SV group, except that had no differential expression of complement component C1q binding, proteoglycan binding, low-density lipoprotein particle binding, and laminin binding. In addition, hemoglobin binding, haptoglobin binding, arachidonic acid binding, Toll-like receptor 4 binding, peptidoglycan binding, and heme binding proteins were also differentially expressed in the PV group. Enrichment in pathway showed that differentially expressed proteins for both the vitiligo stages were involved in complement cascade, clotting cascade, sequestering of ions, and reverse cholesterol transport. However, amb2 integrin signaling and retinoid metabolism pathway were only identified in the progressive stage. The differentially expressed proteins in the biological process were those involved in complement activation, B cell and phagocytosis recognition, and innate immune response of both the vitiligo groups. Differentially expressed proteins related to responses to bacterium and the acute-phase process were detected in PV. The differentially expressed proteins related to cell components included proteins involved in extracellular components such as exosomes, Ig complexes, and the related matrix, in both the vitiligo groups. Functional proteins involved in the acute-phase response, such as the sequestering of ions, reverse lipid transport, and oxygen transport, were markedly highly expressed in the PV group, indicating that these proteins and their molecular functions and pathways may play primary roles in the pathogenesis of vitiligo. Based on the results obtained from the iTRAQ-based proteomic analyses, the four most prominent differentially expressed proteins were reexamined by Western blotting. The two proteins (SERPINA5 and HGFAC) were upregulated in both the stages of vitiligo. SERPINA5 expression was significantly different in both the stages of vitiligo (SV vs. control, P < 0.05; PV vs. control, P < 0.05). In addition, PLCH2 expression was upregulated in the SV (SV vs. control, P < 0.05) and LUZP4 was significantly downregulated in the PV (PV vs. control, P < 0.01) [Figure 1]. Figure 1 The differential expression of four proteins in the different stages of vitiligo and controls. (a) Each column represents one group, and the groups were as follows: controls, patients with stable, and those with progressive patients. Beta-actin was used as the loading control. (b) The gray value ratio of each band compared between the groups (SV or PV compared with control) was used to calculate the significant differences by one-way analysis of variance. *Represents that the difference is statistically significant in PLCH2 (P < 0.05, SV vs. control);† represents that the difference is statistically significant in LUZP4 (P < 0.01, PV vs. control); ‡represents that the difference is statistically significant in SERPINA5 (P < 0.05, SV vs. control and PV vs. control). SV: Stable vitiligo; PV: Progressive vitiligo; HGFAC: Hepatocyte growth factor activator; LUZP4: Leucine zipper protein 4; PLCH2: Phosphoinositide phospholipase C; SERPINA5: Plasma serine protease inhibitor. To better understand the mechanism underlying the pathogenesis of vitiligo, a protein interaction network for the differentially expressed proteins identified in the SV and PV groups was constructed using Cytoscape. The proteins marked with circles and different colors were identified in our analysis, and those marked with boxes are the linker proteins added by the Cytoscape platform [Supplementary Figure 3]. Supplementary Figure 3 Protein interaction network of vitiligo. (a) Protein interaction network in stable vitiligo; (b) protein interaction network of progressive vitiligo. The proteins marked with circles and different colors were identified, and those marked with boxes are the linker proteins added by the Cytoscape platform. Click here for additional data file. Vitiligo is a common chronic acquired disease characterized by depigmentation. Currently, no specific curative therapy and no satisfactory method are available to predict or control the progression of the disease. Proteomics is a feasible approach for large-scale screening of vitiligo-related proteins to elucidate its pathogenesis. The present study employed iTRAQ-based quantitative proteomic tools to identify vitiligo-related proteins in the serum of patients with vitiligo. Disadvantage of this method is that the differentially expressed proteins identified might not fully represent the differentially expressed proteins in the independent sample. However, we generally choose the intersection of different proteins identified in the mixed samples and then selected individual samples to validate the proteomic results by Western blotting experiment. This method has also been proved to be reasonable and feasible in several reports. Our results revealed differentially expressed proteins in the vitiligo samples. Among them, 39 differentially expressed proteins were detected in both the vitiligo groups. Autoimmunity is believed to be the primary cause of vitiligo. In our study, we identified many Ig heavy chain V proteins and Ig chain C proteins that were differentially expressed in SV and PV compared with the controls. We also identified the IgA complex significantly differentially expressed in both the stages of vitiligo compared to the controls from the cell component analysis. Therefore, the increased or decreased plasma levels of Ig heavy chain V or chain C might be potential group proteins requiring further investigation. Our proteomic analysis also showed increased levels of apolipoproteins (i.e., apolipoprotein A1, apolipoprotein A2, and apolipoprotein B) and decreased levels of serum paraoxonase/arylesterase 1 (PON1). These proteins were also analyzed to relate with lipid digestion, mobilization, and transport pathway. Pietrzak et al.[2] reported that lipid metabolism was disrupted in vitiligo-affected children, possibly resulting from disrupted metabolic processes in the adipose tissue as well as oxidative stress. In our study, we found that PON1 levels were decreased in SV but not in PV. This finding indicates that PON1 may decrease when patients are in a stable condition. Zinc-alpha-2-glycoprotein, an essential component of numerous proteins involved in biological defense mechanisms and functioning against oxidative stress, is differentially expressed in patients with SV. Some of these identified proteins are Zn(2+) dependent, such as the plasma protein histidine-rich glycoprotein.[3] Thus, we propose that zinc ion-binding proteins may play a role in the pathogenesis of vitiligo. In addition to the proteins involved in zinc ion binding, some of the identified proteins were involved in calcium ion binding, such as PLCH2 and vitamin D-binding protein. The results of Western blotting test revealed that PLCH2 levels were increased in both the stages of vitiligo, especially in SV. These results showed that proteins with functions related to sequestering calcium ions and reverse cholesterol transport were expressed at markedly high levels in the PV group. There are reports in the literature that polymorphisms in the vitamin D receptor are associated with vitiligo.[4] Clinical trials have also shown that the plasma levels of 25-hydroxy vitamin D and calcium are significantly decreased in patients with vitiligo. Ongoing studies continue to uncover potential roles for the components of the neurosensory system in the skin homeostasis and disease states. In addition, interestingly, proteins involved in other pathways were identified and further verified through Western blotting. SERPINA5 protein, a negative regulator of the Toll pathway, was increased in both SV and PV. It seemed to be associated with micropapillary growth and the invasive phenotype of serious vitiligo that had protease inhibitor-independent activity. The expression and role of serine-type endopeptidase inhibitors in the differentiation of human skin pigmentation remains elusive. Some studies have identified a serine-type protease inhibitor related to palmitoyltransferase that has an effect on melanogenesis.[5] Among other known serine protease inhibitors (SERPINs), the enhanced stability of PAI-1 might play a role in the development of autoimmune disease and the pathophysiology of vitiligo. Moreover, another protein HGFAC was identified and validated in the serum of patients with vitiligo. This protein has serine-type endopeptidase activity and was found to play a role in malignant melanoma progression. Interestingly, LUZP4 levels were found to be decreased in both the vitiligo stages compared to those in the controls. Although LUZP4 was not associated with vitiligo, it has been frequently reported to be activated in melanoma, where it is required for growth.[6] LUZP4 may function to promote the export of mRNAs, which would normally function to export proteins. Thus, it is possible that LUZP4 could also affect melanocyte cell growth. In conclusion, our findings indicated that the autoimmunity proteins, lipid metabolism, oxidative stress proteins (Ig heavy chain V and C, HBB, HBG1, and HBA1), ion-dependent proteins (zinc-alpha-2-glycoprotein, PLCH2, and vitamin D-binding protein), and serine-type inhibitor proteins (increases in SERPINA5 and decreases in LUZP4) might be involved in the pathogenesis of vitiligo. Even though the sample size was small, the differentially expressed proteins that were identified might provide useful information for the diagnosis of early-stage vitiligo prior to the appearance of severe symptoms or for the elucidation of the pathophysiological mechanism. Declaration of patient consent We certify that we have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity. Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website. Financial support and sponsorship This work was supported by grants from the Public Welfare Program, Ministry of Health, China (No. 201202013), and the Innovative Research Team in Universities, Liaoning Bureau of Education (No. LT2012012). Conflicts of interest There are no conflicts of interest.