High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation

Abstract

This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10 ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to mammalian entries in the Swiss-Prot database, using the Mascot search engine. Tryptic peptides originating from aggrecan core protein, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin, thrombospondin-1 (TSP-1), clusterin (CLU), cartilage intermediate layer protein-1 (CILP-1), chondroadherin (CHAD) and matrix metalloproteinases MMP-1 and MMP-3 were detected. Quantitative western blotting confirmed the presence of CILP-1, CLU, MMP-1, MMP-3 and TSP-1. Treatment with IL-1β increased MMP-1, MMP-3 and TSP-1 and decreased the CLU precursor but did not affect CILP-1 and CLU levels. Many of the proteins identified have well-established extracellular matrix functions and are involved in early repair/stress responses in cartilage. This high throughput approach may be used to study the changes that occur in the early stages of osteoarthritis.

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

“…In this part of the review, we focused on proteins identified either directly in the secretome of cartilage explants [12][13][14][15][16][17][18][19][20] or chondrocytes cultures [18,[21][22][23][24][25][26][27][28][29][30][31]), both of which are listed in Table 1. We further complete this list with proteins recently identified by proteomic analysis performed directly on fresh chondrocytes or cartilage tissue [32][33][34][35][36] (sometimes de-cellularized [37]) whereby different locations in the joint were compared, or healthy joint tissues were compared with 6 OA tissues.…”

“…Finally, thrombospondin-1,-3,-4 are all involved in cell-ECM interaction and angiogenesis, and are also secreted by chondrocytes [13,14,17,19,21,24,32,34,35,37]. They are more abundant in the superficial layer of cartilage [34].…”

Biomarkers and proteomic analysis of osteoarthritis

“…Clutterbuck et al adopted a different strategy to investigate the role of inflammatory mediators in a cartilage model system (Clutterbuck et al, 2011). They compared the proteomic profile of cartilage explant culture media when treated under three conditions, medium alone, presence of the inflammatory mediator IL-1β, and IL-1β with the antiinflammatory drug, carprofen.…”

Proteomics in Veterinary Medicine

“…29 An excellent example of how classical histochemistry can be integrated with proteomic techniques is observed in a study focused on equine recurrent uveitis. In a label-free LC-MS/MS analysis of vitreus from uveitis, beside an increase in complement cascade-involved proteins, a significant decrease of proteins involved in Wnt signaling (ie, DKK3 and SFRP2) was recently detected.…”