Characterization of Protein N‐Glycosylation

Although mass spectrometry (MS)‐based protein identification is a straightforward task, the characterization of most posttranslational modifications still represents a challenge. N‐glycosylation with its well known consensus sequence, common core structure, and “universally” active endoglycosidase seems to belong to the easier category. In this chapter, MS methods for the analysis of N‐glycosylated proteins are reviewed. In particular, LC–MS analysis of glycoprotein digests is discussed in detail. The examples included in this chapter illustrate the improved detection sensitivities achieved during the last decade. The characterization of site heterogeneity and of site occupancy is addressed. Low‐energy collision‐induced dissociation (CID) fragmentation of N‐linked glycopeptides and their sodium‐adducts is also described.