High-Resolution Ion Mobility Separations of Isomeric Glycoforms with Variations on the Peptide and Glycan Levels

Glycosylation is a ubiquitous post-translational modification (PTM) that strongly affects the protein folding and function. Glycosylation patterns are impacted by many diseases, making promising biomarkers. Glycans are also the most complex PTMs, exhibiting isomers (linkage, anomers, and those with isomeric moieties). Permuted with localization variants that occur for all PTMs, these produce numerous isomeric glycoforms. Characterizing them by mass spectrometry and ion mobility spectrometry (IMS) has been a challenge. High-definition differential IMS (FAIMS) had robustly disentangled isomeric peptides involving other PTMs but was not evaluated for glycopeptides that featured multilevel isomerism. Here, we apply it to representative mucin glycopeptides with O-linked glycans: three GalNAc localization variants, a pair with alpha/beta GalNAc anomers, and another with GalNAc/GlcNAc isomers. The first two classes were separated baseline with the resolution exceeding previous benchmarks by 10-fold, and the last pair was partly resolved. The recently demonstrated straightforward coupling to ultrahigh-resolution MS and electron-transfer dissociation makes high-definition FAIMS an attractive tool for glycoproteomics.