title: Automated assignments of N- and O-site specific glycosylation with extensive glycan heterogeneity of glycoprotein mixtures.
abstract: Site-specific glycosylation (SSG) of glycoproteins remains a considerable challenge and limits further progress in the areas of proteomics and glycomics. Effective methods require new approaches in sample preparation, detection, and data analysis. While the field has advanced in sample preparation and detection, automated data analysis remains an important goal. A new bioinformatics approach implemented in software called GP Finder automatically distinguishes correct assignments from random matches and complements experimental techniques that are optimal for glycopeptides, including nonspecific proteolysis and high mass resolution liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for multiple N- and O-glycosylation sites, including extensive glycan heterogeneity, was annotated for single proteins and protein mixtures with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide matches and demonstrating the proof-of-concept for a self-consistency scoring algorithm shown to be compliant with the target-decoy approach (TDA). The approach was further applied to a mixture of N-glycoproteins from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein (vLDL) particles.
authors.affiliation: Department of Chemistry, University of California, Davis, California 95616, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Strum
authors.forename: Charles C
authors.initials: CC
authors.lastname: Nwosu
authors.forename: Serenus
authors.initials: S
authors.lastname: Hua
authors.forename: Scott R
authors.initials: SR
authors.lastname: Kronewitter
authors.forename: Richard R
authors.initials: RR
authors.lastname: Seipert
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bachelor
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: An
authors.forename: Carlito B
authors.initials: CB
authors.lastname: Lebrilla
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000032:analysis / Q000378:metabolism*; D010100:Oxygen / Q000032:analysis / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011897:Random Allocation; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D009584:Nitrogen; D010100:Oxygen