期刊
GLYCOCONJUGATE JOURNAL
卷 35, 期 1, 页码 15-29出版社
SPRINGER
DOI: 10.1007/s10719-017-9793-4
关键词
N-glycan; Glycoprotein; Porous graphitised carbon; Liquid chromatography; Mass spectrometry
资金
- Macquarie University Research Excellence Scheme postgraduate scholarship
- Northern Translational Cancer Research Unit through a Cancer Institute NSW competitive student grant
- National eResearch Collaboration Tools and Resources project (NeCTAR) [RT016]
Porous graphitised carbon-liquid chromatography (PGC-LC) has been proven to be a powerful technique for the analysis and characterisation of complex mixtures of isomeric and isobaric glycan structures. Here we evaluate the elution behaviour of N-glycans on PGC-LC and thereby provide the potential of using chromatographic separation properties, together with mass spectrometry (MS) fragmentation, to determine glycan structure assignments more easily. We used previously reported N-glycan structures released from the purified glycoproteins Immunoglobulin G (IgG), Immunoglobulin A (IgA), lactoferrin, alpha 1-acid glycoprotein, Ribonuclease B (RNase B), fetuin and ovalbumin to profile their behaviour on capillary PGC-LC-MS. Over 100 glycan structures were determined by MS/MS, and together with targeted exoglycosidase digestions, created a N-glycan PGC retention library covering a full spectrum of biologically significant N-glycans from pauci mannose to sialylated tetra-antennary classes. The resultant PGC retention library (http://www.glycostore.org/showPgc) incorporates retention times and supporting fragmentation spectra including exoglycosidase digestion products, and provides detailed knowledge on the elution properties of N-glycans by PGC-LC. Consequently, this platform should serve as a valuable resource for facilitating the detailed analysis of the glycosylation of both purified recombinant, and complex mixtures of, glycoproteins using established workflows.
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