Native Mass Spectrometry Meets Glycomics: Resolving Structural Detail and Occupancy of Glycans on Intact Glycoproteins

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.