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 peptidomic analysis highlighted the breakdown products of the fibrinogen alpha and beta chains and cartilage, mainly proteoglycan 4, fibronectins and multiple  collagens characterized by higher number of  post-translational modifications (PTM), such as carbonylations and oxidations (on W, Y, R, K, P and M) as compared with the control subjects, supporting  the involvement of the oxidative stress during inflammatory response.  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 and extrinsic prothrombin and the amplification of the acute phase signaling. The profiling of the JIA-SF peptidome coupled with the new PTM analysis could help to improve the understanding of the mechanisms involved in the amplification of the autoimmune processes during inflammation in JIA disease.

Structural characterization and post-translational modifications (PTM) profiling of the peptidome from the juvenile idiopathic arthritis (JIA) synovial fluid using nanoLC-MS/MS

248th National Meeting (2014)

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