Comparison of Enrichment Methods for Intact N- and
O‑Linked Glycopeptides Using Strong Anion Exchange and Hydrophilic
Interaction Liquid Chromatography
Version 2 2017-10-12, 13:24
Version 1 2017-10-12, 13:20
Posted on 2017-10-12 - 13:24
Heterogeneity of
protein glycosylation poses great challenges for
analysis that is key to understand structure and function of glycoproteins.
Resolving this conundrum requires efficient and specific enrichment
of intact glycopeptides for identification and quantitation. To this
end, hydrophilic interaction chromatography (HILIC) has been commonly
used to enrich intact N- and O-linked glycopeptides. However, its
effectiveness to enrich isobarically labeled glycopeptides remains
unclear. Here, we studied three different enrichment methods for enrichment
of N- and O-linked glycopeptides. It was found that removal of N-glycans
prior to enrichment of O-linked glycopeptides by HILIC improved identification
of O-linked glycopeptides by mass spectrometry. We also compared the
enrichment of intact N- and O-linked glycopeptides using other chromatography
methods and found that using cartridges containing materials for strong
anion exchange (SAX) chromatography increased yield and identification
of N- and O-linked glycopeptides. The enrichment of O-linked glycopeptides
was further improved when a Retain AX cartridge (RAX) was used. In
particular, isobaric tag labeled glycopeptides after C18 desalting
could be readily enriched by SAX and RAX cartridges but not by HILIC
to enable quantitative glycoproteomics. It is anticipated that the
use of SAX and RAX cartridges will facilitate broad applications of
identifications and quantitation of glycoproteins.
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Yang, Weiming; Shah, Punit; Hu, Yingwei; Eshghi, Shadi Toghi; Sun, Shisheng; Liu, Yang; et al. (2017). Comparison of Enrichment Methods for Intact N- and
O‑Linked Glycopeptides Using Strong Anion Exchange and Hydrophilic
Interaction Liquid Chromatography. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.7b03641
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AUTHORS (7)
WY
Weiming Yang
PS
Punit Shah
YH
Yingwei Hu
SE
Shadi Toghi Eshghi
SS
Shisheng Sun
YL
Yang Liu
HZ
Hui Zhang