title: Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry.
abstract: The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Centre , Padualaan 8, 3584 CH Utrecht, The Netherlands.|School of Chemistry and Materials Science, Nanjing Normal University , 1 Weyuan Road, Nanjing, Jiangsu 210023, China.
authors.forename: Guanbo
authors.identifier: 0000-0002-5468-8993
authors.initials: G
authors.lastname: Wang
authors.affiliation: Genmab , Yalelaan 60, 3584 CM Utrecht, The Netherlands.
authors.forename: Rob N
authors.initials: RN
authors.lastname: de Jong
authors.affiliation: Genmab , Yalelaan 60, 3584 CM Utrecht, The Netherlands.
authors.forename: Ewald T J
authors.initials: ETJ
authors.lastname: van den Bremer
authors.affiliation: Genmab , Yalelaan 60, 3584 CM Utrecht, The Netherlands.|Department of Immunohematology and Blood Transfusion, Leiden University Medical Center , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
authors.forename: Paul W H I
authors.initials: PWHI
authors.lastname: Parren
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University , Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Centre , Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Albert J R
authors.identifier: 0000-0002-2405-4404
authors.initials: AJR
authors.lastname: Heck
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000737:chemistry*; D000068598:Data Accuracy; D066246:ErbB Receptors / Q000737:chemistry*; D008970:Molecular Weight; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D066246:ErbB Receptors; D051997:ADP-ribosyl Cyclase 1