Analysis of the Synovial Proteome in Juvenile Idiopathic Arthritis

Background: Juvenile idiopathic arthritis (JIA) comprises a heterogeneous group of chronic, childhood onset, autoimmune diseases with highly variable clinical presentations, outcomes and therapeutic responses. Although clinical evaluation has been a central and important method to pediatric rheumatologists of establishing likely diagnoses and outcomes, laboratory testing for pathogenic biomarkers within joint fluid and peripheral blood samples has been limited. In this paper we present preliminary data of the complex protein profiles within synovial fluid by 2-dimensional electrophoreisis (2DE) methods. Methods: 2DE is a powerful and sensitive technique for separating and analyzing protein mixtures from biological samples. Serial synovial fluid samples, collected from patients displaying a progression in disease, were clarified by centrifugation. Synovial fluid samples were prepared in sample rehydration buffer (8M Urea, 2% CHAPS, 0.5% (v/v) ZOOM carrier ampholytes (Invitrogen) and 0.002% bromophenol blue). The first dimension separation of proteins by isoelectric focusing (based on isoelectric point or pI) was carried out after applying 200 ug (by BCA protein assay) of synovial fluid to immobilized pH gradient (IPG) strips. The second dimension separation (based on molecular weight) was run using denaturing (4-12%) polyacrylamide gel electrophoreisis (SDS-PAGE). The focused proteins were visualized on the completed 2DE gels by colloidal coomassie blue staining and digital images captured by a CCD camera with transilumination. Results: The majority of focused protein spots were within pH range 4 –7. Prominent disease-specific differences in protein expression patterns were observed between samples taken from the same knee in a patient displaying disease progression from monoto pauciarticular JIA. Specific protein spot intensities from both high and low-mass regions of the gel were quantified using the Phoretics 2D software package (Non-linear Dynamics Ltd., UK). Protein spots with significant fold differences in expression were excised from gels, trypsin digested and further characterized by mass spectrometry. Conclusions: The characterization of the synovial proteome from patients with JIA may reveal a small subset of biomarkers/putative therapeutic targets that play a specific role determining the pathophysiological state within the chronically inflamed joint. This large scale validation of differential protein expression profiles will facilitate the prediction of disease susceptibility, assist in diagnosis, further define disease staging, and could allow the selection of individualized therapies or monitoring of treatment responses.