Site-specific glycan heterogeneity characterization by HILIC solid-phase extraction, RPLC fractionation, and capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry.

Reversed-phase chromatographic separation of glycopeptides tends to be dominated by the peptide composition. In contrast, capillary zone electrophoresis separation of glycopeptides is particularly sensitive 'to 'the sialic acid composition of the glycan. In this paper, we combine the two technique's to achieve superior N-glycopeptide analysis. Glycopeptides were first isolated: from a tryptic digest using hydrophilic interaction liquid chromatography (HILIc) solid phase extraction. The glycopeptides were separated using reversed-phase ultra high-performance liquid chromatography (UHPLC) to generate four fractions corresponding to different peptide backbones. Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) was used to analyze the fractions. We applied this method for the analysis of alpha-l-acid glycoprotein (AGP). A total of 268 site-specific N-glycopeptides were detected, representing eight different glycosylation sites from two isomers of AGP. Glycans included tetra-sialic acids with multi N-acetyllactosamine (LacNAc) repeats and unusual pentasialylated terminal sialic acids. Reversed-phase UHPLC coupled with CZE generated, similar to 35% more N-glycopeptides than direct reversed-phase UHPLC-ESI-MS/MS analysis and, similar to 70% more N-glycopeptides than direct CZE-ESI-MS/MS analysis. This approach is a promising tool for global, site-specific glycosylation analysis of highly heterogeneous glycoproteins with mass-limited samples.