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

Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

  • Organism: Homo sapiens, Sus scrofa, Bos taurus
  • Instrument: Velos Plus
  • SpikeIn: No
  • Keywords: Glycomics, Protein Glycosylation, normalisation, LC-MS
Abstract
Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.
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 3/8/19, 10:28 AM
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Flag FileDownloadCreatedListsSmall MoleculesPrecursorsTransitionsReplicates
HLF with ladder_GUnames_2018-08-15_19-59-17.sky.zip (176 KB)2019-03-0864345451
U87MG with ladder_GUnames_2018-08-15_20-02-38.sky.zip (429 KB)2019-03-0891131181181
IgA with ladder_GUnames_2018-08-15_20-00-17.sky.zip (358 KB)2019-03-0881231251251
HNE with ladder_GUnames_2018-08-15_19-59-46.sky.zip (174 KB)2019-03-0863638381
PGM OG with ladder_GUnames_2018-08-15_20-01-34.sky.zip (370 KB)2019-03-0821041061061
IgG with ladder_GUnames_2018-08-15_20-01-05.sky.zip (228 KB)2019-03-0875961611
Fet NG with ladder_GUnames_2018-08-15_19-58-42.sky.zip (229 KB)2019-03-0876063631
CBHI OG with ladder_GUnames_2018-08-15_19-58-20.sky.zip (93 KB)2019-03-0821820201
CBHI NG with ladder_GUnames_2018-08-15_19-57-24.sky.zip (109 KB)2019-03-0832628281
BLF with ladder_GUnames_2018-08-15_19-55-59.sky.zip (302 KB)2019-03-0888385851
Fig3_U87MG_iRT_ladder_2018-08-15_19-50-07.sky.zip (429 KB)2019-03-0891141191191
Fig5_SupFig3_Dextran_Spectral_Library_2018-08-15_19-53-02.sky.zip (40 KB)2019-03-08133181
Fig1_SupFig1_Ladder_tech_reps_2018-08-15_19-48-46.sky.zip (797 KB)2019-03-08111151510
Fig6_2019-01-26_23-48-44.sky.zip (63 MB)2019-03-08811511634212
Fig2_SupFig2_iRT_linear.sky.zip (6 MB)2019-03-08218183216