title: Removal of N-Linked Glycosylations at Acidic pH by PNGase A Facilitates Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of N-Linked Glycoproteins.
abstract: Protein glycosylation is the most frequent post-translational modification and is present on more than 50% of eukaryotic proteins. Glycosylation covers a wide subset of modifications involving many types of complex oligosaccharide structures, making structural analysis of glycoproteins and their glycans challenging for most analytical techniques. Hydrogen/deuterium exchange monitored by mass spectrometry is a sensitive technique for investigation of protein conformational dynamics of complex heterogeneous proteins in solution. N-linked glycoproteins however pose a challenge for HDX-MS. HDX information can typically not be obtained from regions of the glycoprotein that contain the actual N-linked glycan as glycan heterogeneity combined with pepsin digestion yields a large diversity of peptic N-glycosylated peptides that can be difficult to detect. Here, we present a novel HDX-MS workflow for analysis of the conformational dynamics of N-linked glycoproteins that utilizes the enzyme PNGase A for deglycosylation of labeled peptic N-linked glycopeptides at HDX quench conditions, i.e., acidic pH and low temperature. PNGase A-based deglycosylation is thus performed after labeling (post-HDX) and the utility of this approach is demonstrated during analysis of the monoclonal antibody Trastuzumab for which it has been shown that the native conformational dynamics is dependent on the N-linked glycan. In summary, the HDX-MS workflow with integrated PNGase A deglycosylation enables analysis of the native HDX of protein regions containing N-linked glycan sites and should thus significantly improve our ability to study the conformational properties of glycoproteins.
authors.affiliation: Department of Pharmacy, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.
authors.forename: Pernille Foged
authors.initials: PF
authors.lastname: Jensen
authors.affiliation: Department of Pharmacy, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.
authors.forename: Gerard
authors.initials: G
authors.lastname: Comamala
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Morten Beck
authors.identifier: 0000-0002-5220-4297
authors.initials: MB
authors.lastname: Trelle
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Jeppe Buur
authors.initials: JB
authors.lastname: Madsen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.affiliation: Department of Pharmacy, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.
authors.forename: Kasper D
authors.identifier: 0000-0002-6337-5489
authors.initials: KD
authors.lastname: Rand
mesh_terms: D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods; D006020:Glycopeptides / Q000032:analysis* / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D000068878:Trastuzumab / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000068878:Trastuzumab