Application Of Two-dimensional Electrophoresis And Matrix-assisted Laser Desorption-ionization Time Of Flight Mass Spectrometry In Proteomic Analysis Of Synovial Fluid From Knee Osteoarthritis Patients

Objectives: We aimed to analyze the differences in Proteome of synovial fluid(SF) between KOA and non-KOA patients by two-dimensional electrophoresis (2-DE)and matrix-assisted laser desorption-ionization time of flight mass spectrometry(MALDI-TOF-MS). Our primary purpose was to gain insight into the pathogenesis ofKOA and search biomarkers which can be applied in diagnosing preclinical KOA,monitoring the response of joint tissues to therapy and prognosticating the rate of jointdestruction.Methods: The SF samples were collected from12KOA patients and12non-KOA patients with other knee disorders (10of whom had a meniscus injury inthe knee;2had a discoid meniscus in the knee, and all of them exhibited intactarticular cartilage without cartilage defect). All the subjects received arthroscopy inPLA general hospital during July2011to February2012. KOA severity was assessedaccording to the Kellgren-Lawrence radiographic grading criteria. Proteome of thesamples were separated and identified via immobilized pH gradient (IPG). Isoelectricfocusing (IEF) electrophoresis was run as the first dimensional electrophoresis, andthen horizontal SDS-PAGE as the second electrophoresis. After silver staining,images were captured by scanner and then were edited and matched by ImageMaster2D Platinum analysis software. The differentially expressed proteins were identifiedby peptide mass fingerprint (PMF) based on MALDI-TOF-MS and databasesearching. The levels of the proteins which were considered to have potential clinicalsignificance in SF were measured by enzyme-linked immunosorbent assay (ELISA)after identification． Results: A total of31protein spots showed significant differences (p<0.05)between the sample groups, pI of which ranged from3.0to10.0and molecular weightfrom14.0kDa to97.0kDa.25of the31spots (80.6%) were identified as proteins ofinterest. Among them12(48%) were classified into structural proteins,5(20%) intometabolic enzymes,2(8%) into transporters,2(8%) into immunoglobulins and4(16%) into others.20of the25proteins (80%) corresponded to up-regulation and5(20%) to down-regulation in KOA samples. HLA-DR and haptoglobulin was two ofthe proteins up-regulated, which was confirmed by ELISA. Their levels in SF weresignificantly correlated with disease severity.Conclusions:1. Using immobilized pH gradient2-DE, good reproducibility and images could beobtained to separate and identify the proteome in synovial fluid.2. There are some differences between synovial fluid proteome from KOA patientsand that from non-KOA patients3. Some proteins which were found differentially expressed in KOA and non-KOAmight play an important role in disease pathogenesis and could be used asbiomarkers to diagnose and treat KOA in the early stage, monitor curative effectand evaluate prognosis.