Glycosylation is an essential protein modification occurring
on
the majority of extracellular human proteins, with mass spectrometry
(MS) being an indispensable tool for its analysis, that not only determines
glycan compositions, but also the position of the glycan at specific
sites via glycoproteomics. However, glycans are complex branching
structures with monosaccharides interconnected in a variety of biologically
relevant linkages, isomeric properties that are invisible when the
readout is mass alone. Here, we developed an LC-MS/MS-based workflow
for determining glycopeptide isomer ratios. Making use of isomerically
defined glyco(peptide) standards, we observed marked differences in
fragmentation behavior between isomer pairs when subjected to collision
energy gradients, specifically in terms of the galactosylation/sialylation
branching and linkage. These behaviors were developed into component
variables that allowed for relative quantification of isomerism within
mixtures. Importantly, at least for small peptides, the isomer quantification
appeared to be largely independent from the peptide portion of the
conjugate, allowing a broad application of the method.