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.
Chemical deglycosylation O-glycoproteins O-glycoproteome liquid chromatography-mass spectrometry proteomics
human glycophorin A
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Ishii-Karakasa, I., Iwase, H., Hotta, K. (1997) Structural determination of the O-linked sialyl oligosaccharides liberated from fetuin with endo-α-N-acetylgalactosaminidase-S by HPLC analysis and 600-MHz 1H-NMR spectroscopy. Eur. J. Biochem. 247, 709–715PubMedCrossRefGoogle Scholar
Umemoto, J., Bhavanandan, V.P., Davidson, E.A. (1977) Purification and properties of an endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J. Biol. Chem. 252, 8609–8614PubMedGoogle Scholar
Patel, T.P., Parekh, R.B. (1994) Release of oligosaccharides from glycoproteins by hydrazinolysis. Methods Enzymol. 230, 57–66PubMedCrossRefGoogle Scholar
Gerken, T.A., Gupta, R., Jentoft, N. (1992) A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins. Biochemistry31, 639–648PubMedCrossRefGoogle Scholar
Rademaker, G.J., Pergantis, S.A., Blok-Tip, L.A., Langridge, J.I., et al. (1998) Mass spectrometric determination of the sites of O-glycan attachment with low picomolar sensitivity. Anal. Biochem. 257, 149–160PubMedCrossRefGoogle Scholar
Hanisch, F.-G., Jovanovic, M., Peter-Katalinic, J. (2001) Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments. Anal. Biochem. 290, 47–59PubMedCrossRefGoogle Scholar
Hong, J.C., Kim, Y.S. (2000) Alkali-catalyzed beta-elimination of periodate-oxidized glycans: a novel method of chemical deglycosylation of mucin gene products in paraffin-embedded sections. Glycoconj. J. 17, 691–703PubMedCrossRefGoogle Scholar
Hanisch, F.-G., Teitz, S., Schwientek, T., Müller, S. (2009) Chemical de-O-glycosylation of glycoproteins for application in LC-based proteomics. Proteomics9, 710–719PubMedCrossRefGoogle Scholar
Müller, S., Hanisch, F.G. (2002) Recombinant MUC1 probe authentically reflects cell-specific O-glycosylation profiles of endogenous breast cancer mucin. J. Biol. Chem. 277, 26103–26112PubMedCrossRefGoogle Scholar
Engelmann, K., Kinlough, C.L., Müller, S., Razawi, H., et al. (2005) Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. Glycobiology15, 1111–1124PubMedCrossRefGoogle Scholar