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Development of peptidomic assays for discovery of endogenously processed peptides from the healthy and juvenile idiopathic arthritis human synovial fluid (981.9)
Author(s) -
Clement Cristina,
Santambrogio Laura
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.981.9
Subject(s) - chemistry , orbitrap , synovial fluid , proteome , arthritis , tandem mass spectrometry , computational biology , mass spectrometry , osteoarthritis , biochemistry , immunology , medicine , biology , pathology , chromatography , alternative medicine
Juvenile idiopathic arthritis (JIA) is the most common chronic condition seen by pediatric rheumatologists. We and others hypothesized that joint destruction could be partly due to an adaptive immune response mediated by antibodies, and that the characterization of the peptidome (together with the degradome) found in arthritic synovial fluid (SF) (from children with JIA) will help to advance the present studies and shed new molecular mechanisms on the pathogenesis of JIA. Herein, we present the development of peptidomics assays coupled with high resolution nanoLC‐MS/MS on LTQ/Orbitrap Velos mass spectrometer aimed to characterize the endogenously processed peptides from the SF of children with JIA and control patients. The SF peptidome was fractionated by ultrafiltration and peptides with MW<5 kDa were sequenced by HCD/ETD nanoLC Orbitrap‐ESI‐MS/MS or CID nanoLC LTQ‐ESI‐MS/MS. The MS/MS spectra were analyzed with two searching engines, Mascot and PEAKS, against SwissProt database. The peptidome analysis highlighted the breakdown products of the fibrinogen alpha and beta chains and cartilage, mainly proteoglycan 4, fibronectins and multiple collagen subtypes (I, II, III, IV, V, among others). Ingenuity Pathway Analysis (IPA) revealed that the SF from JIA patients is enriched with acute phase reactants, as well as structural components having more than 80% of the proteome connected into the pathways characterizing the inflammatory phenotype: activation of the complement, coagulation systems, intrinsic snd extrinsic prothrombin and the amplification of the acute phase signaling. We proposed that the peptidome biomarkers found in the JIA‐SF could help to elucidate the mechanism for the amplification of the autoimmune process during inflammation. Moreover, the peptidomic assays described herein could be applied to other biological samples of biomedical interest and further used to investigate the proteases (the degradome) responsible for regulating the bioactive peptides.