Nano reversed phase versus nano hydrophilic interaction liquid chromatography on a chip in the analysis of hemopexin glycopeptides

- NanoRP-LC and HILIC were used for the separation of the glycopeptides of hemopexin.
- NanoHILIC provided higher selectivity based on glycan type compared to nanoRP-LC.
- NanoRP-LC method provided higher sensitivity compared to the nanoHILIC method.
- NanoHILIC method partially separated fucose linkage isomers on the A2G2 glycoforms.

Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.