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Glycoproteins are inherently heterogeneous and therefore resolving structures in their entirety remains a major challenge in structural biology. Native mass spectrometry has transformed our ability to study glycoproteins, and despite advances in high-resolution instrumentation, there are comparatively a few studies demonstrating its potential with data largely limited to an overall measure of monosaccharide composition for all glycans across glycosylation sites for a given protein. Clearly, these readouts lack glycan topology information, namely, monosaccharide linkage and glycan branching. To address this deficiency, we developed a new approach that joins native mass spectrometry with glycan exoglycosidase sequencing, the combination of which provides remarkable glycoprotein structural details. We show how N-glycan branching, terminal fucosylation, LacNAc extensions, and N- and O-glycan occupancy (i.e., total number of glycans) can be directly characterized on intact glycoproteins with minimal sample preparation. Taken together, native exoglycosidase sequencing mass spectrometry (NES-MS) notably improves our ability to characterize protein glycosylation, addressing a significant need in structural biology that will enable new routes to understand glycoprotein function.

Original publication




Journal article


Anal Chem

Publication Date





10435 - 10443


Glycomics, Glycoproteins, Glycosylation, Mass Spectrometry, Polysaccharides