Characterization of glycosylation on the glycopeptide level can be challenging with traditional peptide mapping methods that employ reversed-phase liquid chromatography coupled with tandem mass spectrometry (RPLC- MS/MS or MSE). These methods typically employ collision induced dissociation (CID) as the fragmentation technique, which has limitations for site specific analysis of labile modifications. The use of Electron Capture Dissociation (ECD) enables glycan moieties to be retained on the peptide backbone, and allow the site to be confidently assigned using the resulting fragment ions. In this study, we demonstrate the utility of ECD on the SELECT SERIES Cyclic IMS instrument for unambiguous assignment of glycosylation site locations for 11 O- glycopeptide species of ENBREL (etanercept).
ECD fragmentation generates critical site-specific O-glycosylation information Improved sensitivity, resolution, and IMS capabilities of the SELECT SERIES Cyclic IMS instrument provide comprehensive analytical tools to unravel this complex set of biotherapeutic attributes.
Recombinant biotherapeutics produced in mammalian systems generally contain a variety of post-translational modifications (PTMs), one of the most complex being glycosylation. Glycosylation plays a critical role in stability, activity, and immunogenicity for biotherapeutics,1 and must be clearly characterized and monitored. Unlike N- glycosylation, where there is a consensus sequence1 for occupation of the asparagine site, any exposed serine or threonine site can potentially be O-glycosylated if presented to the glycosyltransferases and glycosidases in the proper 3D structural motif. This means they can be unpredictable and challenging to characterize without prior structural knowledge.
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