Utilisation of a dextran ladder to standardise PGC-LC-MS-based glycomics
Ashwood C, Pratt B, MacLean B, PackerNH. Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics
- Organism: Homo sapiens, Sus scrofa, Bos taurus
- Instrument: Velos Plus
Glycomics, Protein Glycosylation, normalisation, LC-MS
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 for 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 thus built a library of over 300 PGC-separated glycan structures with normalised 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 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 of these efforts, system-independent assignment of glycan structure based only on precursor mass and glucose unit value can be achieved which was previously not possible using PGC-based LC-MS.
Development of a workflow for normalisation of glycomics experiments performed with porous graphitised carbon LC-MS.
N- and O-Glycans released from purified glycoprotein standards, mixed with partially-hydrolysed dextran.
Created on 8/17/18 9:22 AM