title: Glyco-analytical multispecific proteolysis (Glyco-AMP): a simple method for detailed and quantitative Glycoproteomic characterization.
abstract: Despite recent advances, site-specific profiling of protein glycosylation remains a significant analytical challenge for conventional proteomic methodology. To alleviate the issue, we propose glyco-analytical multispecific proteolysis (Glyco-AMP) as a strategy for glycoproteomic characterization. Glyco-AMP consists of rapid, in-solution digestion of an analyte glycoprotein (or glycoprotein mixture) by a multispecific protease (or protease cocktail). Resulting glycopeptides are chromatographically separated by isomer-specific porous graphitized carbon nano-LC, quantified by high-resolution MS, and structurally elucidated by MS/MS. To demonstrate the consistency and customizability of Glyco-AMP methodology, the glyco-analytical performances of multispecific proteases subtilisin, pronase, and proteinase K were characterized in terms of quantitative accuracy, sensitivity, and digestion kinetics. Glyco-AMP was shown be effective on glycoprotein mixtures as well as glycoproteins with multiple glycosylation sites, providing detailed, quantitative, site- and structure-specific information about protein glycosylation.
authors.affiliation: Asia Glycomics Reference Site , Daejeon 305-764, South Korea.
authors.forename: Serenus
authors.initials: S
authors.lastname: Hua
authors.forename: Chloe Y
authors.initials: CY
authors.lastname: Hu
authors.forename: Bum Jin
authors.initials: BJ
authors.lastname: Kim
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Totten
authors.forename: Myung Jin
authors.initials: MJ
authors.lastname: Oh
authors.forename: Nayoung
authors.initials: N
authors.lastname: Yun
authors.forename: Charles C
authors.initials: CC
authors.lastname: Nwosu
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
authors.forename: Carlito B
authors.initials: CB
authors.lastname: Lebrilla
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: An
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000737:chemistry*; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D040901:Proteomics; D012260:Ribonucleases / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D011134:Polysaccharides; D012260:Ribonucleases; C029600:ribonuclease B; D010447:Peptide Hydrolases