Age-related changes in articular cartilage lead to extracellular matrix (ECM) degeneration and potentially the development of inflammatory osteoarthritis (OA). OA is characterised by increased expression and activity of pro-inflammatory cytokines, chemokines, catabolic mediators and ECM degrading enzymes. During LC-MS/MS analysis, high abundance of proteins of the ECM in the secretome prevented identification of differentially released low abundance protein with potential involvement in cartilage inflammation and ECM degradation. Here we describe the use of two methods, (cetylpyridinium chloride (CPC) precipitation and conacanavalin A (Con A) chromatography), to bind and deplete negatively-charged glycosylated side chains of high abundance ECM proteins and proteoglycans with the objective of uncovering and identifying lower abundance proteins, which would subsequently be quantified by western blotting. Articular cartilage explant cultures were established from equine metacarpophalangeal joints (tissues derived from animals euthanased for reasons other than research) and incubated (37°C, 5% CO2) with the pro-inflammatory cytokine IL-1β (10 ng/ml) for 6 days. CPC (5%) was added (3 mg CPC/mg PG), samples were incubated (RT, 30 minutes), and centrifuged (13,000 rpm, 10 minutes). Con A was added (0.5 ml slurry per 200 µl sample) and centrifuged in spin columns (3,300 rpm, 4 minutes). An amaZon speed ETD (Bruker) was used for nanoLC-MS/MS to analyse the principle protein components of each sample. For protein identifications the NCBInr database was searched using Mascot, only peptides with individual ions scores with a P<0.05 or greater were accepted. Silver stained SDS-PAGE protein profiles confirmed that depletion techniques removed many high abundance proteins, leaving a simplified secretome profile. Sixty proteins were identified in undepleted IL-1β treated samples. After Con A depletion 39 proteins were identified. One ECM constituent, HPLP-1, was enriched by Con A depletion with a mascot score of 796. CPC precipitation identified 34 proteins. Both CPC precipitation and Con A chromatography reduced mascot scores for many proteins, especially small proteoglycans including decorin and fibromodulin. Four additional intracellular proteins were reliably identified after depletion steps, including ferritin heavy chain 1 (score 250) and manganese superoxide dismutase (score 260). In conclusion, whilst depletion methods removed many abundant ECM proteins they were ineffective in identifying a greater range of inflammatory related proteins and few additional low abundance proteins were detected. Such proteins may have been co-extracted through binding with selectively-removed ECM components, or interacted with CPC/Con A.