title: In-depth glycoproteomic characterisation of grape berry vacuolar invertase using a combination of mass spectrometry-based approaches.
abstract: Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins.
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Agnès
authors.initials: A
authors.lastname: Hovasse
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Tchilabalo Dilezitoko
authors.initials: TD
authors.lastname: Alayi
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.affiliation: Laboratoire d'Oenologie et Chimie Appliquée, URVVC EA 4707, Université de Reims Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.
authors.forename: Richard
authors.initials: R
authors.lastname: Marchal
authors.affiliation: Laboratoire d'Oenologie et Chimie Appliquée, URVVC EA 4707, Université de Reims Champagne-Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Jégou
authors.affiliation: BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France. Electronic address: christine.schaeffer@unistra.fr.
authors.forename: Christine
authors.initials: C
authors.lastname: Schaeffer-Reiss
mesh_terms: D000595:Amino Acid Sequence; D006020:Glycopeptides / Q000032:analysis*; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry / Q000379:methods*; D027843:Vitis / Q000201:enzymology*; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006020:Glycopeptides; D043324:beta-Fructofuranosidase
keywords: Deglycosylation; Glycopeptide; Grape; Mass spectrometry; N-Glycosylation; Vacuolar invertase; Wine