Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics

Porous graphitised carbon (PGC) based chromatographic separation of glycans achieves high-resolution separation of glycan mixtures released from glycoproteins, including structurally similar isomers. While there is some understanding of glycan separation on PGC, system-independent retention values have not been established. Using hydrolysed dextran as an internal standard, and Skyline software for post-acquisition normalisation, retention time and glycan peak area variation of replicate injections of glycan mixtures was significantly reduced. Normalisation of retention time to the dextran ladder allowed assignment of system-independent retention values, values that are applicable to all PGC-based separations regardless of chromatographic system. We have built a library of over 300 PGC-separated glycan structures with assigned normalised glucose unit (GU) PGC retention values. To further define the mechanism of glycan separation with PGC, we identified predictive models for the chromatographic effects resulting from addition and/or removal of core-fucosylation and bisecting GlcNAc based on the PGC normalised retention time library. A dextran ladder spectral library was also built to ensure correct retention time assignment of the internal standard added to glycan mixtures. Using the spectral matching feature in Skyline, isomeric discrimination between O-mannosylated glycans and the glucose-based dextran ladder was achieved. As a result, system-independent automated assignment of glycan structure based on precursor mass and glucose unit value, using a glycan structure reference library, can be achieved using PGC-LC-MS.