title: Comparative analysis of novel autoantibody isotypes against citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)(542-556) peptide in serum from Taiwanese females with rheumatoid arthritis, primary Sjögren's syndrome and secondary Sjögren's syndrome in rheumatoid arthritis.
abstract: The purpose of this study was to discover and validate inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) as novel biomarkers, and evaluate autoantibody isotypes against an unmodified and citrullinated ITIH3(542-556) peptide among Taiwanese female patients with rheumatoid arthritis (RA), primary Sjögren's syndrome (pSS), secondary Sjögren's syndrome in rheumatoid arthritis (RA-sSS), and healthy controls (HCs). We used concanavalin A (Con A) affinity chromatography, 1-D SDS-PAGE, and label-free nano-LC-MS/MS to screen biomarker candidates (serum-derived Con A-captured proteins) and then identify PTMs of validated biomarkers (serum proteins) using pooled serum from 7 RA-sSS female patients and 7 age-matched HCs (the discovery set). Furthermore, the protein level and autoantibody isotype analyses were further validated against individual serum from 18 HCs, 18 RA, 18 pSS, and 18 RA-sSS patients (the validation set). Con A-bound ITIH3 was identified and validated as the only differentially expressed protein, which was elevated. Additionally, 2 novel PTMs in ITIH3 were identified and included citrullination at arginine-(546) and arginine-(556), and hexosamine at tryptophan-(558). Further, concentrations of anti-citrullinatd-ITIH3(542-556) peptide autoantibodies significantly increased in patients with RA, pSS, and RA-sSS compared to HCs. Especially, autoantibody IgM against the citrullinated-ITIH3(542-556) peptide showed better diagnostic performance in discriminating both RA versus pSS and pSS versus RA-sSS.
  
  
  
  By using comparative proteomic analysis of serum samples, the current study discovered and validates differentially expressed Con A-bound ITIH3 as a potential biomarker for secondary Sjögren's syndrome (SS) in rheumatoid arthritis (RA) patients and healthy controls (HCs). Besides, hexosamine and citrullination on ITIH3 were further identified. Through analyzing autoantibody isotypes against the citrullinated ITIH3 peptide, patients with RA, primary SS, and RA-secondary SS, and HCs can be further discriminated. The current strategy can be applied for identifying potential diagnostic and pathologic markers for autoimmune diseases.
authors.affiliation: Proteomics Research Center, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Chen-Chung
authors.initials: CC
authors.lastname: Liao
authors.affiliation: Division of Allergy-Immunology-Rheumatology, Department of Internal Medicine, Min-Sheng General Hospital, Taoyuan 330, Taiwan; Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taipei Medical University-Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan.
authors.forename: Pei-Lun
authors.initials: PL
authors.lastname: Chou
authors.affiliation: Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Chao-Wen
authors.initials: CW
authors.lastname: Cheng
authors.affiliation: Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taipei Medical University-Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan.
authors.forename: Yu-Sheng
authors.initials: YS
authors.lastname: Chang
authors.affiliation: Department of Laboratory Medicine, Taipei Medical University, -Shuang-Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan.
authors.forename: Wei-Ming
authors.initials: WM
authors.lastname: Chi
authors.affiliation: Division of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Taipei Medical University-Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan.
authors.forename: Kai-Leun
authors.initials: KL
authors.lastname: Tsai
authors.affiliation: Department of Biotechnology and Animal Science, National Ilan University, Ilan 26047, Taiwan.
authors.forename: Wei-Jung
authors.initials: WJ
authors.lastname: Chen
authors.affiliation: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Ting-Shuan
authors.initials: TS
authors.lastname: Kung
authors.affiliation: Department of Laboratory Medicine, Taipei Medical University, -Shuang-Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan.
authors.forename: Chih-Chun
authors.initials: CC
authors.lastname: Tai
authors.affiliation: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan.
authors.forename: Kuan-Wei
authors.initials: KW
authors.lastname: Lee
authors.affiliation: PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan.
authors.forename: You-Chia
authors.initials: YC
authors.lastname: Chen
authors.affiliation: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan; PhD Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11031, Taiwan. Electronic address: cylin@tmu.edu.tw.
authors.forename: Ching-Yu
authors.initials: CY
authors.lastname: Lin
mesh_terms: D000328:Adult; D000510:Alpha-Globulins / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000175:diagnosis / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D001798:Blood Proteins / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D005260:Female; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000097:blood / Q000276:immunology; D008875:Middle Aged; D012859:Sjogren's Syndrome / Q000175:diagnosis / Q000276:immunology*; D013624:Taiwan
chemical_list: D000510:Alpha-Globulins; D001323:Autoantibodies; D001798:Blood Proteins; D007132:Immunoglobulin Isotypes; D002956:Citrulline; C011877:inter-alpha-inhibitor
keywords: Autoantibody isotypes; Citrullination; Primary Sjögren's syndrome; Rheumatoid arthritis; Secondary Sjögren's syndrome in rheumatoid arthritis; Serum