Mass spectrometric profiling ofO-linked glycans released directly from glycoproteins in gels using in-gel reductive β-elimination

Abstract

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.