Glycoconjugate glycans play vital roles in a wide range of physiological and pathological processes, such as embryogenesis, host-pathogen interactions, inflammation, and tumorigenesis. In the pharmaceutical industry, glycosylation is a key determinant for the solubility, stability, efficacy, and immunogenicity of recombinant monoclonal antibody therapeutics.

Conclusions• In this application note, we show that EED can generate far richer structural information than CID for elucidation of the glycan sequence (topology) and linkage configurations. Presently, EED of glycans has only been demonstrated on MRMS instruments, whose high mass resolving power and mass accuracy are critical for accurate peak assignments. Coupling of EED MS/MS with on-line LC separation allows characterization of glycan mixtures with isomeric structures. Finally, one can take advantage of the complexity of the glycan EED spectra to improve the confidence in fragment assignments by considering the contextual features of the peak of interest. The workflow presented here provides a powerful tool for facile