Glycopeptide mapping of glycoproteins presents a highly effective technique that can be used to elucidate both domain and peptide-specific glycosylation. In this work, we have demonstrated the use of an ACQUITY UPLC Glycoprotein BEH Amide 300A 1.7 μm Column to obtain HILIC separations of glycopeptides that complement the chromatographic information afforded by a reversed phase separation. In addition, our results indicate that these HILIC separations provide exemplary peak capacity in comparison to other commercially available amide column technologies. That the HILIC separation is MS-compatible means that information-rich data can be readily acquired to characterize a glycopeptide map.
For instance, this work shows that it can be a relatively straightforward exercise to characterize multidomain protein glycosylation, such as the Fc and Fab domain glycosylation of cetuximab. Combined with other recently developed strategies, such as HILIC subunit mapping and GlycoWorks RapiFluor-MS released N-glycan analyses, glycopeptide mapping with the ACQUITY UPLC Glycoprotein BEH Amide Column shows significant promise for facilitating the characterization of protein glycosylation to unprecedented levels of detail.