To capitalize on the proficiency of some of the new mucinases, like SmE, for cleaving glycoproteins, high performance MS/MS methods are needed to characterize the resulting ensembles of glycopeptides. Traditionally, collisionally activation dissociation (CAD) has been used to identify glycan compositions of glycopeptides through the production of B, C, Y, and Z glycosidic fragments. In addition, CAD produces b/y ions that provide peptide sequence information, but the ability of CAD to localize glycosites is impeded owing to the tendency of CAD to cleave labile PTMs from the peptides. Other MS/MS methods have proven more successful for glycosite localization. Electron transfer dissociation (ETD) generates c/z sequence ions that retain PTMs, including glycans, allowing mapping of glycosites of numerous O-glycoproteins. Combining ETD with supplemental collisional activation enhances the production of fragment ions, and one of these hybrid methods, EThcD (electron transfer higher collision energy dissociation), is a powerhouse for O-glycosite localization and characterization of multi-charged glycopeptides. With the ability to localize glycans and offer detailed information about their structures, EThcD is widely used for O-glycoproteomics, as demonstrated for probing the glycoproteome of human serum, investigating glycoproteins in cerebrospinal fluid, deciphering glycosylation of antibodies and their components, and analyzing the O-glycan profiles of individual glycoproteins. The natural complexity of glycans creates challenges even for the most versatile MS/MS methods like EThcD. Glycans may exist in multiple structural arrangements even if the absolute composition is the same, as exemplified by the structure diversity of a sialylated core 1 glycan, composed of a single Nacetylgalactosamine (GalNAc), galactose (Gal), and sialic acid (Neu5Ac). The three saccharides can be linked sequentially or both the Gal and the Neu5Ac can be attached to the GalNAc. Additionally, the linkage between the Neu5Ac and Gal may be α2,3 or α2,6, thus encoding an additional level of structural detail.