title: Nano-LC-MS/MS of glycopeptides produced by nonspecific proteolysis enables rapid and extensive site-specific glycosylation determination.
abstract: Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, United States.
authors.forename: John W
authors.initials: JW
authors.lastname: Froehlich
authors.forename: Mariana
authors.initials: M
authors.lastname: Barboza
authors.forename: Caroline
authors.initials: C
authors.lastname: Chu
authors.forename: Larry A
authors.initials: LA
authors.lastname: Lerno
authors.forename: Brian H
authors.initials: BH
authors.lastname: Clowers
authors.forename: Angela M
authors.initials: AM
authors.lastname: Zivkovic
authors.forename: J Bruce
authors.initials: JB
authors.lastname: German
authors.forename: Carlito B
authors.initials: CB
authors.lastname: Lebrilla
mesh_terms: D000818:Animals; D002645:Chickens; D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000032:analysis; D059748:Proteolysis; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides