How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides

Z. Darula, J. Sherman, K. F. Medzihradszky

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ∼25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higherenergy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Volume11
Issue number7
DOIs
Publication statusPublished - Jul 2012

Fingerprint

Mucin-1
Glycopeptides
Mucins
Chromatography
Peptides
Affinity Chromatography
Proteins
Electrons
Glycosylation
Affinity chromatography
Workflow
Glycoside Hydrolases
Ion Exchange Chromatography
Serum
Static Electricity
Oligosaccharides
Hydrophobic and Hydrophilic Interactions
Lectins
Sugars
Proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

Cite this

How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides. / Darula, Z.; Sherman, J.; Medzihradszky, K. F.

In: Molecular and Cellular Proteomics, Vol. 11, No. 7, 07.2012.

Research output: Contribution to journalArticle

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