title: A procedure for the analysis of site-specific and structure-specific fucosylation in alpha-1-antitrypsin.
abstract: A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.|Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong.
authors.forename: Haidi
authors.initials: H
authors.lastname: Yin
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.
authors.forename: Zhijing
authors.initials: Z
authors.lastname: Tan
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA.
authors.forename: Mingrui
authors.initials: M
authors.lastname: An
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
authors.forename: Shiyue
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA.
authors.forename: Yehia
authors.initials: Y
authors.lastname: Mechref
authors.affiliation: Department of Surgery, University of Michigan Medical Center, Ann Arbor, MI, USA. dmlubman@umich.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
mesh_terms: D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002853:Chromatography, Liquid / Q000379:methods; D005643:Fucose / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D000515:alpha 1-Antitrypsin; D005643:Fucose; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Alpha-1-antitrypsin; Antennary fucosylation; Core fucosylation; Glycan structure; Liquid chromatography-mass spectrometry