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Improved online LC-MS/MS identification of O-glycosites by EThcD fragmentation, chemoenzymatic reaction, and SPE enrichment.

Authors
  • Yang, Shuang1
  • Wang, Yan2
  • Mann, Matthew1
  • Wang, Qiong3
  • Tian, E4
  • Zhang, Liping4
  • Cipollo, John F3
  • Ten Hagen, Kelly G4
  • Tabak, Lawrence A5
  • 1 Biological Chemistry Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA.
  • 2 Mass Spectrometry Facility, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA.
  • 3 Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.
  • 4 Developmental Glycobiology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA.
  • 5 Biological Chemistry Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892, USA. [email protected]
Type
Published Article
Journal
Glycoconjugate Journal
Publisher
Springer-Verlag
Publication Date
Apr 01, 2021
Volume
38
Issue
2
Pages
145–156
Identifiers
DOI: 10.1007/s10719-020-09952-w
PMID: 33068214
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

O-glycosylation is a highly diverse and complex form of protein post-translational modification. Mucin-type O-glycosylation is initiated by the transfer of N-acetyl-galactosamine (GalNAc) to the hydroxyl group of serine, threonine and tyrosine residues through catalysis by a family of glycosyltransferases, the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (E.C. 2.4.1.41) that are conserved across metazoans. In the last decade, structural characterization of glycosylation has substantially advanced due to the development of analytical methods and advances in mass spectrometry. However, O-glycosite mapping remains challenging since mucin-type O-glycans are densely packed, often protecting proteins from cleavage by proteases. Adding to the complexity is the fact that a given glycosite can be modified by different glycans, resulting in an array of glycoforms rising from one glycosite. In this study, we investigated conditions of solid phase extraction (SPE) enrichment, protease digestion, and Electron-transfer/Higher Energy Collision Dissociation (EThcD) fragmentation to optimize identification of O-glycosites in densely glycosylated proteins. Our results revealed that anion-exchange stationary phase is sufficient for glycopeptide enrichment; however, the use of a hydrophobic-containing sorbent was detrimental to the binding of polar-hydrophilic glycopeptides. Different proteases can be employed for enhancing coverage of O-glycosites, while derivatization of negatively charged amino acids or sialic acids would enhance the identification of a short O-glycopeptides. Using a longer than normal electron transfer dissociation (ETD) reaction time, we obtained enhanced coverage of peptide bonds that facilitated the localization of O-glycosites. O-glycosite mapping strategy via proteases, cut-off filtration and solid-phase chemoenzymatic processing. Glycopeptides are enriched by SPE column, followed by release of N-glycans, collection of higher MW O-glycopeptides via MW cut-off filter, O-glycopeptide release via O-protease, and finally detected by LC-MS/MS using EThcD.

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