Application Of ITRAQ And SWATH In Proteomic Analysis Of Synovial Fluid In Knee Osteoarthritis

Objectives:We aimed to analyze the differences in Proteome of synovial fluid (SF) between KOA, RA and meniscus injury patients by isobaric tags for relative and absolute quantitation (iTRAQ) and sequential windowed acquisition of all theoretical fragment ions (SWATH). Our primary purpose was to gain insight into the pathogenesis of KOA and search biomarkers which can be applied in diagnosing preclinical KOA, monitoring the response of joint tissues to therapy and prognosticating the rate of joint destruction.Methods:1. The SF samples were collected from 10 KOA patients who received a arthroplasty and 10 meniscus injury patients who received a arthroscopy (all of them exhibited intact articular cartilage without cartilage defect under arthroscopy). All the subjects received the surgery in PLA general hospital during June 2014 to September 2014. KOA severity was assessed according to the Kellgren-Lawrence radiographic grading criteria and all the KOA patients in the study group were classified into KL 4 grade. Proteome of the SF samples were analyzed and compared by iTRAQ technique to scan for the differently expressed proteins preliminarily, and GO analysis, COG analysis and pathway enrichment were performed for these differently expressed proteins.2. The SF samples were collected from 10 KOA patients and 10 RA patients who received a arthroplasty and 10 meniscus injury patients who received a arthroscopy (all of them exhibited intact articular cartilage without cartilage defect under arthroscopy). All the KOA patients and RA patients were classified into KL 4 grade according to the Kellgren-Lawrence radiographic grading criteria. Proteome of the SF samples were analyzed and compared by SWATH technique to scan for the specific proteins which differently expressed in the KOA. GO analysis and pathway enrichment were performed for differently expressed proteins. The levels of the proteins which were considered to have potential clinical significance in SF were measured by enzyme-linked immunosorbent assay (ELISA) after identification.Results:1. Comparing the proteome of SF from KOA patients and meniscus injury patients by iTRAQ, we identified a total of 71 proteins which showed statistically significant differences.31 of the 71 proteins corresponded to up-regulation and 40 to down-regulation in KOA samples. GO analysis showed that the cellular component of differently expressed proteins mainly derived from extracellular matrix (ECM), membrane, organelle, and macromolecular complex; the molecular function of these protein included binding, transporter activity, catalytic activity, and enzyme regulator activity; the biological process these differently expressed proteins participated in mainly included response to stimulus, regulation of biological process, and metabolic process. Pathway enrichment showed that most differently expressed proteins participated in complement and coagulation cascades as well as ECM-receptor interaction.2. Comparing the proteome of SF from KOA patients, RA patients and meniscus injury patients by SWATH, we obtained results as following: ①KOA vs meniscus injury:a total of 131 proteins showed statistically significant differences, of which 93 proteins corresponded to up-regulation and 38 proteins to down-regulation in KOA samples. The differences in expressions of complement Clr and fatty acid-binding protein (FABP) were the most significant. ②RA vs meniscus injury:a total of 185 proteins showed statistically significant differences, of which 152 proteins corresponded to up-regulation and 33 proteins to down-regulation in RA samples. ③KOA vs RA:a total of 139 proteins showed statistically significant differences, of which 22 proteins corresponded to up-regulation and 117 proteins to down-regulation in KOA samples.28 proteins expressed differently only in the KOA, of which 17 proteins corresponded to up-regulation and 11 proteins to down-regulation, and Dickkopf-related protein was one of the proteins up-regulated in KOA specifically.8 proteins expressed contrarily in KOA and RA, of which 3 proteins corresponded to up-regulation in KOA and to down-regulation in RA,5 proteins corresponded to down-regulation in KOA and to up-regulation in RA. GO analysis with respect to the 131 differently expressed proteins showed that the cellular component of differently expressed proteins mainly derived from ECM, membrane, cytoplasm, secretory granule, and lipoprotein complex; the molecular function of these protein included enzyme activity, enzyme inhibitor activity, binding, transporter, and regulation activity; the biological process these differently expressed proteins participated in mainly included acute inflammatory response to wound, activation of complement, immune response, response to stimulus, regulation of apoptosis, and homeostasis. Pathway enrichment showed that most differently expressed proteins participated in complement and coagulation cascades, ECM-receptor interaction, and peroxisome proliferator activated receptor (PPAR) signaling pathway. ELISA indicated that complement Clr and Dickkopf-related protein were significantly up-regulated in KOA, whereas fatty acid-binding protein was significantly down-regulated in KOA. The levels of complement Clr and Dickkopf-related protein in SF were positively correlated with disease severity, whereas the level of fatty acid-binding protein in SF were negatively correlated with disease severity.Conclusions:1. As non-specific proteomic technique, iTRAQ and SWATH were characterized by high sensitivity, excellent quantification effect and producibility. Furthermore, They had advantages in the coverage rate of protein detection and the throughput of analysis.2. Some proteins which were found differentially expressed in KOA and non-KOA might play an important role in disease pathogenesis and could be used as biomarkers to diagnose and treat KOA in the early stage, monitor curative effect and evaluate prognosis.