Isotypes of autoantibodies against differentially expressed novel malondialdehyde-modified peptide adducts in serum of Taiwanese women with rheumatoid arthritis.

This study identified and validated four differentially expressed novel malondialdehyde (MDA)-modified peptide adducts and evaluated autoantibodies against native and MDA-modified peptides among Taiwanese women patients with rheumatoid arthritis (RA), osteoarthritis (OA) and healthy controls (HCs). Ig kappa chain C region76-99, alpha-1-antitrypsin284-298, alpha-2-macroglobulin824-841, and apolipoprotein B-1004022-4040 exhibiting 2-fold differences in relative modification ratios were identified by concanavalin A (Con A) affinity chromatography, 1D SDS-PAGE, in-gel digestion, nano-LC/MS/MS and nano-LC/MS using pooled serum-derived Con A-captured proteins from 9 RA and 9 age-matched HCs. Furthermore, the levels of proteins, serum MDA, and MDA-modified protein adducts were further validated against individual serum from 20 RA and 20 HCs, and autoantibodies against native and their MDA-modified peptides used 45 RA, 30 OA and 45 HCs. Levels of serum MDA and MDA-modified protein adducts were significantly higher in RA than HCs but protein levels were not significantly different. Serum Igs G and M against MDA-modified peptides showed better diagnostic performance in differentiating among patients with RA, OA and HCs, with an area under the receiver operating characteristic curve of 0.96-0.98, sensitivity of 88.9%-97.8%, and specificity of 88.9%-100%. Autoantibodies against MDA-modified epitopes become useful clinical biomarkers for RA. BIOLOGICAL SIGNIFICANCE: By using a label-free relative quantitative proteomic analysis of concanavalin A (Con A)-bound serum samples, the current study discovered and validated malondialdehyde (MDA)-modified peptide adducts as novel biomarkers for differentiating between rheumatoid arthritis (RA) patients and healthy controls (HCs). In addition, the serum levels of MDA, proteins, and MDA-modified protein adducts as well as the MDA modification of proteins were determined. Isotypes of autoantibodies against MDA-modified peptide adducts can be used as serological biomarkers for further discriminating among RA patients, osteoarthritis patients and HCs. This strategy can become the basis for identifying potential diagnostic and pathological biomarkers for RA.

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.

UNLABELLED: 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. SIGNIFICANCE: 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.