Estimation of the identity of proteolytic aggrecan fragments using PAGE migration and Western immunoblot.

Objective: To develop calculation models, using Western immunoblot, as a tool for the estimation of proteolytic human aggrecan fragment identity. Method. Seven human aggrecan fragments (calibrators), purified by CsCl gradient centrifugation and identified by Western immunoblot of N- and C-terminals, were used to develop calculation models. The models were used for identification of unknown aggrecan fragments each having one of their N- or C-terminals identified. Results: The calibrator molecular weights (Mw) from sodium dodecyl sulfate (SDS)-gels (m), the Mw of amino acids (a) and the Mw of their carbohydrate substitution (g) were expressed as K = m/(a + g), or as K = 1.085 m/(a + g) when compensation for the G1 domain was required. Using these models together with average K-values, 12 out of the 17 immuno-detected aggrecan fragments were calculated to a known protease cleavage site, while five were identified to domain levels. Conclusions: With six neoepitope antibodies together with antibodies against the G1- and G3-domain it was possible to predict the identity of several proteolytic fragments from different regions within the aggrecan monomer. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.