Yamagiwa et al. demonstrated a five fold increase in the expression of 18 protein spots including haptoglobin among different synovial fluid samples from OA patients using Y-27632 mw 2 DE platform. In another study, 135 proteins were identified from synovial fluid and 18 of them were shown to be differentially expressed in OA patients. Pro teins identified to be elevated in OA included alpha 1 mi croglobulin, apolipoprotein E, complement component 3, haptoglobin, orosomucoid 1 and group specific compo nent. A method of en dogenous profiling of peptides from OA synovial fluid that resulted in identification of 40 proteins was described by Kamphorst et al. in 2007. In a recent study, abnor mally high levels of complement components were shown in OA synovial fluid. Sohn et al.
identified 108 pro teins from OA synovial fluid and found that only 36% of them were known to be in the Inhibitors,Modulators,Libraries plasmaserum. Sixty six proteins, involved in acute phase response, comple ment and coagulation pathways were reported to be differ entially expressed between healthy and OA synovial fluid in a recent study by Ritter et al. A summary of previous proteomic studies on OA synovial fluid is pro vided in Table 1. Most of these investigations were carried out using low resolution mass spectrometers and with minimal fractionation of the samples, which limited the depth of coverage. In this study, we carried out a compre hensive cataloging of proteins from OA synovial fluid by including multiple fractionation methods followed by high resolution mass spectrometry analysis.
Results and discussion Identification of proteins from OA synovial fluid Synovial fluid from five OA Inhibitors,Modulators,Libraries patients was pooled and the abundant proteins were depleted using Human MARS 6 column. The resulting sample was then subjected to Inhibitors,Modulators,Libraries mul tiple fractionation methods Inhibitors,Modulators,Libraries SDS PAGE at the protein level and SCX and OFFGEL at the peptide level to reduce the complexity of the sample. In addition, lectin enrichment strategy was employed to enrich Inhibitors,Modulators,Libraries glycoproteins using a mix ture of three different lectins wheat germ agglutinin, concanavalin A and jacalin. These lectins have different binding specificities and thereby permit enrichment of a broader coverage of glycoproteins. The lectin enriched frac tions were subjected to SDS PAGE and SCX fractionation. All of these fractions were analyzed on a Fourier transform LTQ Orbitrap Velos mass spectrometer. The workflow il lustrating the steps involved in the proteomic analysis of OA synovial fluid is shown in Figure 1. 124,380 peptide spectrum matches generated from the MG132 clinical mass spectrometric analysis of 112 fractions of depleted and lectin enriched OA synovial fluid resulted in the iden tification of 5,544 peptides corresponding to 677 proteins.