Proteominer facilitates high‐throughput proteomic analysis of low abundance proteins in the cartilage secretome (LB39)

Abstract

Proteomics is increasingly used for biomarker identification. We have developed high‐throughput mass spectrometric (MS) methods to identify proteins in the secretome of articular cartilage explants, which we are utilizing as an in vitro model of joint inflammation. However, the identification of lower abundance proteins of potentially higher biological significance in this model is hampered by the presence of highly abundant glycoproteins in the secretome. Development of protein depletion techniques that allow the identification of low abundance proteins is important for novel biomarker discovery, especially in the context of identifying pathophysiologically relevant biomarkers of joint inflammation and cartilage degradation. Proteominer protein enrichment kits decrease the dynamic range of complex biological samples, allowing improved analytical coverage of the proteome. The objective of this study was to apply high‐throughput MS techniques following proteominer enrichment to expand the range of proteins that can be identified in the secretome of cartilage exposed to proinflammatory cytokines such as IL‐1β. Explant cultures were established in DMEM + 2% Pen/Strep, using metacarpophalangeal joints of horses. Explants were either untreated controls or treated with 10 ng/ml IL‐1β. After incubating for 6 days at 37⁰C in a humidified environment (95% air, 5% CO2) proteins in culture supernatants were concentrated ~24 fold using Centriplus YM‐3 and Centricon‐3 (3000 MWCO) centrifugal filter units. Concentrated samples were processed with a Small Capacity Proteominer™ Protein Enrichment Kit (Bio‐Rad). After elution, samples were reduced, alkylated, acetone precipitated and trypsin digested overnight, before digested peptides were applied to C18 spin columns, followed by nanoLC‐MS/MS analysis on an amaZon speed ETD. Extracellular matrix proteins identified in untreated control secretome included cartilage oligomeric matrix protein (COMP), fibromodulin and fibronectin. Macrophage migration inhibitory factor (MIF), serum amyloid A protein (SAA) and Heat shock 70 kDa protein 1‐like (HSP70) were identified in explants exposed to IL‐1β. Identification of new low abundance proteins in this model will facilitate a greater understanding of inflammatory processes occurring within cartilage.Grant Funding Source: Supported by Biotechnology and Biological Sciences Research Council (BBSRC) (Contract grant number: BB/G018030/1) and the European Commission Framework 7 program (EU FP7; HEALTH.2012.2.4.5–2, project number 305815).