There are two main glycosylation types in mammals: N-glycosylation occurs at the carboxamido nitrogen on asparagine residues (Asn) of secreted/membrane proteins within the consensus sequence Asn-Xaa-Thr/Ser, where Xaa is any amino acid except for proline.86 The second main type of glycosylation is O-glycosylation, in which sugar residues attach to serine and threonine residues (Ser, Thr) or, to a much lesser extent to hydroxyproline and hydroxylysine.87 The latter two are particularly abundant in collagens and add an additional level of regulation to collagen biosynthesis. If both present, O-glycosylation occurs after N-glycosylation. Moreover, O-glycosylation is not restricted to secreted proteins and to date, no consensus sequences have been identified for this PTM.88 ECM proteins may be extensively modified by addition of N- and O-linked large and repetitive glycosaminoglycans (GAGs) and shorter and diverse N- and O-linked oligosaccharides. Aberrant glycosylation can lead to pathological abnormalities and disease. In the last decade, proteomics has emerged as a powerful platform to characterize glycosylation profiles of ECM proteins, including the cardiovascular field.52,58,89 There are two major strategies that can be used to study glycoproteins by MS (Figure 4).