title: How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides.
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 higher-energy 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.
authors.affiliation: Proteomics Research Group, Biological Research Center of Hungarian Academy of Sciences, Szeged, H-6701, Szeged, POB 521, Hungary.
authors.forename: Z
authors.initials: Z
authors.lastname: Darula
authors.forename: J
authors.initials: J
authors.lastname: Sherman
authors.forename: K F
authors.initials: KF
authors.lastname: Medzihradszky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D054794:Glycomics / Q000379:methods*; D006020:Glycopeptides / Q000097:blood*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D018396:Mucin-1; D009844:Oligosaccharides; D010455:Peptides; D006026:Glycoside Hydrolases