Protein glycosylation is widely considered the most dynamic, compositionally complex, and structurally diverse post-translational modification (PTM). For this reason, analytical researchers are often interested in uncovering facile, simultaneous means of PTM identification and structural assignment. Capillary electrophoresis (CE) routinely demonstrates the ability to separate glycoconjugate isomers in the liquid phase but is hampered by difficulties in deciphering conformational information. A significant advancement in ion mobility (IM) technology, the recently developed High-Resolution Demultiplexing (HRDm) technique may serve to remedy these shortcomings by offering improvements in instrument sensitivity and gas-phase resolution. In this study, we hypothesize coupling CE with HRDm-IM analyses will facilitate exponential improvements in detailing the conformational complexity of glycosylated proteins. We tested this hypothesis by coupling CE with this high-resolution IM technology and systematically evaluating a collection of glycoprotein standards (Bovine Fetuin, Asialofetuin) via a sheath flow-coupled CE-IMS platform (Agilent 7100 CE, Agilent 6560 IM-QTOF). Digested proteins were analyzed with and without glycopeptide enrichment in both standard and HRDm modes; endoglycosidase enzymes were employed to aid in structural assignment. Whereas competitive high-resolution chromatography failed to retain or separate glycopeptide isomers, CE enabled near baseline resolution of asialo-/sialoglycopeptides across the migration window. Ion mobility analyses without HRDm revealed poorly-resolved instances of peak broadening and shouldering, indicating the existence of multiple conformations in the gas phase. Utilizing HRDm, we achieved R&gt;150 in the mobility dimension, reproducibly separating these distinct isomeric species. Significantly, while CE enabled the separation of up to 4 isomers per unique glycopeptide in the liquid phase, HRDm revealed as many as 3 distinct conformations for each separated peak. True identities were assigned through endoglycosidase digestion, serving to reveal the conformational complexity inherent to glycoproteins. This novel CE-HRDm-IM workflow enables exponential improvements in the number of identifiable glycopeptide isomers and provides a unique path towards comprehensive characterization of these active biomolecules.

Uncovering glycoprotein compositional and structural heterogeneity through capillary electrophoresis-ion mobility mass spectrometry

ACS Fall 2021

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