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Chemical De-O-glycosylation of Glycoproteins for Applications in LC-Based Proteomics

  • Franz-Georg HanischEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 753)

Abstract

This paper describes a cyclic on-column procedure for the sequential degradation of complex O-glycans on proteins by periodate oxidation of sugars and cleavage of oxidation products by elimination. Glycoproteins are immobilized to alkali-stable, reversed-phase Poros 20 beads, desialylated by treatment with dilute trifluoroacetic acid, and de-O-glycosylated by two degradation cycles before the eluted apoproteins are digested with trypsin for analysis by liquid chromatography electrospray ionization-mass spectrometry. Even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration, or racemization of lysine and arginine residues. The protocol is also applicable on gel-immobilized glycoproteins after 1D or 2D gel electrophoresis. Conversion of O-glycoproteins into their corresponding apoproteins results in facilitated accessibility of tryptic cleavage sites, increases the numbers of peptide fragments, and accordingly enhances protein coverage and identification rates within the subproteome of mucin-type O-glycoproteins. The protocol is suitable for automatization, but due to partial elution from the Poros 20 columns it is not recommended for applications on the glycopeptide level.

Key words

Chemical deglycosylation O-glycoproteins O-glycoproteome liquid chromatography-mass spectrometry proteomics 

Abbreviations

HGA

human glycophorin A

TFMSA

trifluoromethanesulfonic acid

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institute of Biochemistry II, Medical Faculty, and Center for Molecular Medicine CologneUniversity of CologneKölnGermany

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