title: Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.
abstract: A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.
authors.affiliation: Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University School of Medicine , Boston, Massachusetts 02118, United States.
authors.forename: Kshitij
authors.initials: K
authors.lastname: Khatri
authors.forename: Gregory O
authors.initials: GO
authors.lastname: Staples
authors.forename: Nancy
authors.initials: N
authors.lastname: Leymarie
authors.forename: Deborah R
authors.initials: DR
authors.lastname: Leon
authors.forename: Lilla
authors.initials: L
authors.lastname: Turiák
authors.forename: Yu
authors.initials: Y
authors.lastname: Huang
authors.forename: Shun
authors.initials: S
authors.lastname: Yip
authors.forename: Han
authors.initials: H
authors.lastname: Hu
authors.forename: Christian F
authors.initials: CF
authors.lastname: Heckendorf
authors.forename: Joseph
authors.initials: J
authors.lastname: Zaia
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D009961:Orosomucoid / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics
chemical_list: D006023:Glycoproteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D009961:Orosomucoid
keywords: CAD; Glycopeptides; HILIC; tandem MS