Differential proteomic analysis of synovial fluid from rheumatoid arthritis and osteoarthritis patients

Abstract

Background: Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis.

Results: We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ≥3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis.

Conclusions: We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis.Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

Figure 1

A schematic workflow illustrating the steps involved in the differential analysis of RA and OA synovial fluid proteome. Proteins from RA and OA synovial fluid were extracted and depleted to remove the 14 most abundant proteins using multiple affinity removal system, Human-14. The depleted protein from RA and OA were then digested with trypsin and labeled with iTRAQ reagents, 117 and 116 respectively. The labeled samples were pooled and subjected to fractionation using strong cation exchange chromatography. The fractions were then analyzed on a LTQ-Orbitrap Velos mass spectrometer. The MS/MS data obtained was searched against Human RefSeq 50 database using Sequest and Mascot search algorithms. Validation of the iTRAQ quantitation data was carried out using multiple reaction monitoring and Western blot.

Figure 2

Gene Ontology-based classification of differentially expressed proteins identified from RA and OA synovial fluid. (A) Subcellular localization (B) Biological processes.

Figure 3

Representative MS/MS spectra of peptides from novel proteins upregulated in RA and OA synovial fluid. Novel proteins upregulated in RA: (A) Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ) (9.8-fold), (B) Fibrinogen-like protein 2 (FGL2) (3.5-fold). Novel proteins upregulated in OA: (C) CD5 antigen-like precursor (CD5L) (3.3-fold), (D) Soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D) (3.3-fold).

Figure 4

Validation of CAPG by MRM assay and Western blot. A) Box and whisker’s plot shows the relative abundance of CAPG in RA compared to OA synovial fluid. B) MRM peak traces of the peptide, QAALQVAEGFISR (z = +2, m/z = 695.38) from CAPG (RT: Retention time). C) Western blot analysis of CAPG overexpression in RA synovial fluid (n = 10) when compared to OA synovial fluid (n = 10) (Pooled: RA = 10 and OA = 10; R-RA, O-OA).

Figure 5

Glycolytic pathway enriched from GeneSpring analysis. Proteins upregulated in RA are highlighted in the glycolysis pathway image.