Elsevier

Journal of Proteomics

Volume 75, Issue 10, 6 June 2012, Pages 2869-2878
Journal of Proteomics

Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC–MALDI TOF/TOF

https://doi.org/10.1016/j.jprot.2011.12.042Get rights and content

Abstract

The purpose of this study was to identify those proteins relatively more abundant in the synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA) and osteoarthritis (OA) using high performance liquid chromatography coupled to mass spectrometry. 20 individual SF samples from each disease were pooled into two groups (RA and OA) to reduce the contribution of extreme individual values. Prior to the proteomic analysis, samples were immunodepleted from the top 20 most abundant plasma proteins, to enrich the lower-abundance protein fractions. Then, they were subjected to protein size fractioning and in-gel digestion, followed by reversed-phase peptide separation in a nano-LC system and subsequent peptide identification by MALDI-TOF/TOF. This strategy led to the identification of 136 different proteins in SF, which is the largest number of SF proteins described up to date by proteomics.

A relative quantification of the proteins between RA and OA was carried out by spectral counting analysis. In RA, our results show a greater relative abundance of proteins related to complement activation, inflammation and the immune response, such as the major matrix metalloproteinases and several neutrophil-related proteins. In OA, we detected an increase in proteins involved in the formation and remodeling of the extracellular matrix (ECM), such as fibronectin, kininogen-1, cartilage acidic protein 1 and cartilage oligomeric matrix protein. The results obtained for MMP-1, BGH3, fibronectin and gelsolin were verified by immunoblotting analyses. Some of the novel proteins identified in this work might be relevant not only for increasing knowledge on the etiopathogenesis of RA and OA processes, but also as putative disease biomarkers, as their presence in SF is a prior step to their dilution in serum. This article is part of a Special Issue entitled: Proteomics: The clinical link.

Graphical abstract

Highlights

► Synovial fluid immunodepletion of the 20 most abundant plasma proteins. ► The largest number of different proteins reported up to date in SF by proteomics. ► Relative quantification of RA and OA by nanoLC–MALDI-MS/MS and spectral counting. ► Identification of a novel RA SF proteomic signature. ► Identification of a novel OA SF proteomic signature.

Introduction

Synovial fluid (SF) is a serum filtrate located in the joint, where it also receives protein contributions from the surrounding tissues, articular cartilage, synovial membrane and bone [1]. In a healthy joint, articular cartilage and SF cushion the bones and allow them to move easily. Disorders causing destruction of the joint, such as osteoarthritis (OA) and rheumatoid arthritis (RA), are the most common rheumatic pathologies. Although both diseases result in the destruction of articular cartilage, they have very different etiopathogeneses [2], [3]. RA is an autoimmune disease that correlates with a strong inflammation that is responsible for cartilage destruction, mediated by the activation of macrophages and B and T lymphocytes. OA is characterized by alterations in metabolism and cell signaling, and by an imbalance of redox mechanisms leading to degradation of cartilage and surrounding tissues through the action of activated matrix metalloproteinases (MMPs) [4], [5]. The identification of a biomarker or a panel of biomarkers related to the OA process would be of great clinical importance not only for the early diagnosis of the disease, but also for its treatment and the development of experimental and clinical therapeutic trials and promoting a deeper understanding of the disease. Because SF is in direct contact with the affected tissues, cartilage, synovial membrane and bone, it could be an excellent source for discovery of biomarkers [6]. Furthermore, the identification of a differential proteomic signature of these processes will increase our understanding of their underlying molecular basis.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS) has been shown to be a powerful tool for protein profiling in biological fluids and, in recent years, has been widely used for large-scale identification of complex protein mixtures [7]. The high dynamic range in the protein content of body fluids, such as serum, plasma, urine, or synovial fluid, creates the need for the preliminary steps of depleting highly abundant proteins and multi-fractioning the sample [8]. Furthermore, recent improvements in the robustness and mass accuracy of LC–MS platforms make label-free quantitative methods an attractive alternative to such classical labeling methods as isobaric labeling such as iTRAQ, 18O, and others [9]. We have designed a label-free relative quantitative approach using spectral counting, a well-documented label free quantitative technique [10] to identify SF proteins, and we have compared the differential protein profiles between RA and OA. Among the different label-free quantitative proteomics strategies, which essentially use the relationship between sampling statistics and protein abundance [11], spectral counting has proven its utility for profiling complex proteomes [12]. The information about the proteins identified in this work would help us to understand the etiopathogenesis of RA and OA at the molecular level. Moreover, these proteins might also hold an important value as joint-specific disease biomarkers.

Section snippets

Patients

Patients with RA and OA selected for this study were diagnosed following the criteria determined by the American College of Rheumatology (ACR, 1988). The mean age of the RA subjects was 56 ± 10 years old (n = 20), while that of the OA subjects was 73 ± 8 years old (n = 20). All SF samples were collected after informed consent from knee joints by arthrocentesis in the Rheumatology Department at Hospital Universitario de A Coruña, following our institutional regulations and procedures for sample

Handling and immunodepletion of SF samples for proteomic analysis

Synovial fluid is a potential compartment for joint disease biomarkers, as it is derived directly from the site where the pathology occurs. Consequently, many proteomic strategies in the rheumatology field have been designed to identify putative biomarkers in SF, and then try to validate them in serum [6].

SF can be quite easily obtained from knee joints by aseptical aspiration, avoiding blood contamination. Due to its high viscosity, hyaluronidase treatment is a recommended procedure to digest

Conclusions

Protein profiling of complex biological samples has improved in the recent years, largely due to the development of the liquid chromatography coupled to mass spectrometry technique (LC–MS). In this study, we semi-quantitatively analyzed the protein content of knee SF, comparing samples from two highly prevalent pathological conditions affecting articular joints, RA and OA. We demonstrated that the LC–MALDI-TOF/TOF technique is suitable for the ongoing efforts to discover early indicators for RA

Acknowledgments

We thank the ISCIII Networked Proteomics Platform (ProteoRed) and its members for support and helpful discussions, Nieves Domenech for antibody donations and Sara Relaño for technical help. This study was supported by grants from the Instituto de Salud Carlos III (CIBER BBN CB06-01-0040 and FIS-PI 08/2028), with participation of funds from FEDER (European Community); and from Xunta de Galicia (PGIDIT06PXIB916358PR, 10CSA916058PR). P. F.-P. and C.R.-R. are supported by ISCIII.

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