Macquarie U - Dextran ladder to standardise PGC-LC-MS-based glycomics

Utilisation of a dextran ladder to standardise PGC-LC-MS-based glycomics

  • Organism: Homo sapiens, Sus scrofa, Bos taurus
  • Instrument: Velos Plus
  • SpikeIn: No
  • Keywords: Glycomics, Protein Glycosylation, normalisation, LC-MS
Abstract
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.
Experiment Description
Development of a workflow for normalisation of glycomics experiments performed with porous graphitised carbon LC-MS.
Sample Description
N- and O-Glycans released from purified glycoprotein standards, mixed with partially-hydrolysed dextran.
Created on 8/17/18 9:22 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedListsPeptidesSmall MoleculesPrecursorsTransitionsReplicates
Fig2_SupFig2_Skyline_iRT_linear_2018-08-15_23-04-52.sky.zip (1 MB)2018-08-239130131314
U87MG with ladder_GUnames_2018-08-15_20-02-38.sky.zip (429 KB)2018-08-23901131181181
IgA with ladder_GUnames_2018-08-15_20-00-17.sky.zip (358 KB)2018-08-23801231251251
HNE with ladder_GUnames_2018-08-15_19-59-46.sky.zip (174 KB)2018-08-23603638381
PGM OG with ladder_GUnames_2018-08-15_20-01-34.sky.zip (370 KB)2018-08-23201041061061
IgG with ladder_GUnames_2018-08-15_20-01-05.sky.zip (228 KB)2018-08-23705961611
HLF with ladder_GUnames_2018-08-15_19-59-17.sky.zip (176 KB)2018-08-23604345451
Fet NG with ladder_GUnames_2018-08-15_19-58-42.sky.zip (229 KB)2018-08-23706063631
CBHI OG with ladder_GUnames_2018-08-15_19-58-20.sky.zip (93 KB)2018-08-23201820201
CBHI NG with ladder_GUnames_2018-08-15_19-57-24.sky.zip (109 KB)2018-08-23302628281
BLF with ladder_GUnames_2018-08-15_19-55-59.sky.zip (302 KB)2018-08-23808385851
Fig3_U87MG_iRT_ladder_2018-08-15_19-50-07.sky.zip (429 KB)2018-08-23901141191191
Fig2_SupFig2_Skyline_iRT_2018-08-15_19-46-49.sky.zip (1 MB)2018-08-239130131314
Fig5_SupFig3_Dextran_Spectral_Library_2018-08-15_19-53-02.sky.zip (40 KB)2018-08-231033181
Fig1_SupFig1_Ladder_tech_reps_2018-08-15_19-48-46.sky.zip (797 KB)2018-08-231011151510