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Proteomic Analysis of Human Osteoarthritis Synovial Fluid

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Proteomic Analysis of Human Osteoarthritis Synovial Fluid Balakrishnan et al. Clinical Proteomics 2014, 11:6 http://www.clinicalproteomicsjournal.com/content/11/1/6 CLINICAL PROTEOMICS RESEARCH Open Access Proteomic analysis of human osteoarthritis synovial fluid Lavanya Balakrishnan1,2, Raja Sekhar Nirujogi1,3, Sartaj Ahmad1,4, Mitali Bhattacharjee1,5, Srikanth S Manda1,3, Santosh Renuse1,5, Dhanashree S Kelkar1,5, Yashwanth Subbannayya1,6, Rajesh Raju1, Renu Goel1,2, Joji Kurian Thomas1,5, Navjyot Kaur7, Mukesh Dhillon7, Shantal Gupta Tankala7, Ramesh Jois8, Vivek Vasdev9, YL Ramachandra2, Nandini A Sahasrabuddhe1, TS Keshava Prasad1,3,4, Sujatha Mohan10, Harsha Gowda1, Subramanian Shankar7* and Akhilesh Pandey11,12,13,14* Abstract Background: Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. Results: We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. Conclusions: We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis. Keywords: Body fluid, Cartilage, Joint destruction, Glycosylation Background of disability among the aging population. The two im- Osteoarthritis (OA) is a degenerative joint disorder char- portant risk factors for developing OA are obesity and acterized by articular cartilage damage, formation of age [2]. Despite the high prevalence of OA, its mec- osteophytes and subchondral bone cysts, thickened sub- hanism of pathogenesis still remains unclear [3]. The chondral plate, inflammation and neovascularisation of diagnosis of OA can be made based on structural abnor- synovial membrane [1]. OA is one of the leading causes malities or symptoms resulting from these abnormalities. While OA is evident radiologically in most of the elderly * Correspondence: [email protected]; [email protected] population, only 10% are symptomatic and exhibit a 7Department of Internal Medicine, Armed Forces Medical College, Pune, Maharashtra 411040, India measurable limitation of function. Further, radiographs 11McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, may be normal in early disease owing to lack of sensitiv- 733 N. Broadway, BRB 527, Baltimore, MD 21205, USA ity in visualizing minimal cartilage loss [4]. Thus, the Full list of author information is available at the end of the article © 2014 Balakrishnan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Balakrishnan et al. Clinical Proteomics 2014, 11:6 Page 2 of 13 http://www.clinicalproteomicsjournal.com/content/11/1/6 diagnostic tools that are currently in use have their own previous proteomic studies on OA synovial fluid is pro- limitations and provide an inaccurate assessment of dis- vided in Table 1. Most of these investigations were carried ease progression [5]. Finally, the drugs currently used for out using low resolution mass spectrometers and with the treatment of OA are aimed at reducing pain and do minimal fractionation of the samples, which limited the not possess any disease modifying activity [6]. depth of coverage. In this study, we carried out a compre- Studying the synovial fluid proteome should yield a hensive cataloging of proteins from OA synovial fluid by higher concentration of potential biomarkers than serum including multiple fractionation methods followed by high or plasma, as the synovial fluid is in direct physical contact resolution mass spectrometry analysis. with the synovium, ligament, meniscus, joint capsule and bone [7]. Alterations in the structure and metabolism of Results and discussion any of these tissues during disease should be reflected as al- Identification of proteins from OA synovial fluid terations in the composition of the synovial fluid proteome. Synovial fluid from five OA patients was pooled and the Therefore, the synovial fluid proteome has the potential to abundant proteins were depleted using Human MARS-6 indicatetheseverityandprogressionofthedisease[8]. column. The resulting sample was then subjected to mul- Advances in proteomic technologies have facilitated exten- tiple fractionation methods - SDS-PAGE at the protein level sive proteomic characterization of several body fluids and SCX and OFFGEL at the peptide level - to reduce the [9-15]. A detailed molecular characterization of the synovial complexity of the sample. In addition, lectin enrichment fluid could identify proteins associated with pathogenesis, strategy was employed to enrich glycoproteins using a mix- which can be developed as markers for evaluation of the ture of three different lectins - wheat germ agglutinin, disease in early stages and its progression. concanavalin A and jacalin. These lectins have different Yamagiwa et al. demonstrated a five-fold increase in binding specificities and thereby permit enrichment of a the expression of 18 protein spots including haptoglobin broader coverage of glycoproteins. The lectin enriched frac- among different synovial fluid samples from OA patients tions were subjected to SDS-PAGE and SCX fractionation. using 2-DE platform [16]. In another study, 135 proteins All of these fractions were analyzed on a Fourier transform were identified from synovial fluid and 18 of them were LTQ-Orbitrap Velos mass spectrometer. The workflow il- shown to be differentially expressed in OA patients. Pro- lustrating the steps involved in the proteomic analysis of teins identified to be elevated in OA included alpha 1- mi- OA synovial fluid is shown in Figure 1. croglobulin, apolipoprotein E, complement component 3, 124,380 peptide spectrum matches generated from the haptoglobin, orosomucoid 1 and group specific compo- mass spectrometric analysis of 112 fractions of depleted nent (vitamin D binding protein) [3]. A method of en- and lectin-enriched OA synovial fluid resulted in the iden- dogenous profiling of peptides from OA synovial fluid that tification of 5,544 peptides corresponding to 677 proteins. resulted in identification of 40 proteins was described by The number of proteins identified from the depleted and Kamphorst et al. in 2007 [17]. In a recent study, abnor- lectin-enriched fractions are summarized in Additional file mally high levels of complement components were shown 1A and 1B, respectively. Of the 300 lectin-enriched pro- in OA synovial fluid [18]. Sohn et al. identified 108 pro- teins identified, 171 proteins were already known to be teins from OA synovial fluid and found that only 36% of glycosylated from the data available in Human Protein them were known to be in the plasma/serum [19]. Sixty Reference Database (HPRD) [22,23]. The complete list of six proteins, involved in acute phase response, comple- all proteins and peptides identified in our study are pro- ment and coagulation pathways were reported to be differ- vided in Additional files 2 and 3, respectively. The relative entially expressed between healthy and OA synovial fluid abundance of the 25 most abundant proteins identified is in a recent study by Ritter et al [20]. A summary of provided in Additional file 4. Table 1 A summary of proteomic studies published on healthy and osteoarthritis synovial fluid Synovial fluid used Method Mass spectrometer Number of Publication (Healthy/Osteoarthritis) used proteins identified 1 Healthy/Osteoarthritis In-gel digestion LCQ DECA XP 135 Gobezie, R et al., 2007 [3] 2 Osteoarthritis Depletion of albumin & IgG, IEF, XCT Ultra Ion trap 108 Sohn, DH et al., 2012 [19] In-gel digestion 3 Healthy/Osteoarthritis 2D-DIGE - 66 Ritter, SY et al., 2013 [20] 4 Healthy/Osteoarthritis Ultrafiltration and solid phase extraction
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