title: The Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics.
abstract: In-depth site-specific investigations of protein glycosylation are the basis for understanding the biological function of glycoproteins. Mass spectrometry-based N- and O-glycopeptide analyses enable determination of the glycosylation site, site occupancy, as well as glycan varieties present on a particular site. However, the depth of information is highly dependent on the applied analytical tools, including glycopeptide fragmentation regimes and automated data analysis. Here, we used a small set of synthetic disialylated, biantennary N-glycopeptides to systematically tune Q-TOF instrument parameters towards optimal energy stepping collision induced dissociation (CID) of glycopeptides. A linear dependency of m/z-ratio and optimal fragmentation energy was found, showing that with increasing m/z-ratio, more energy is required for glycopeptide fragmentation. Based on these optimized fragmentation parameters, a method combining lower- and higher-energy CID was developed, allowing the online acquisition of glycan and peptide-specific fragments within a single tandem MS experiment. We validated this method analyzing a set of human immunoglobulins (IgA1+2, sIgA, IgG1+2, IgE, IgD, IgM) as well as bovine fetuin. These optimized fragmentation parameters also enabled software-assisted glycopeptide assignment of both N- and O-glycopeptides including information about the most abundant glycan compositions, peptide sequence and putative structures. Twenty-six out of 30 N-glycopeptides and four out of five O-glycopeptides carrying >110 different glycoforms could be identified by this optimized LC-ESI tandem MS method with minimal user input. The Q-TOF based glycopeptide analysis platform presented here opens the way to a range of different applications in glycoproteomics research as well as biopharmaceutical development and quality control.
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Hinneburg
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Stavenhagen
authors.affiliation: Bruker Daltonik GmbH, Bremen, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Schweiger-Hufnagel
authors.affiliation: Bruker Daltonik GmbH, Bremen, Germany.
authors.forename: Stuart
authors.initials: S
authors.lastname: Pengelley
authors.affiliation: Bruker Daltonik GmbH, Bremen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Jabs
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Seeberger
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424, Potsdam, Germany.
authors.forename: Daniel Varón
authors.initials: DV
authors.lastname: Silva
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands.|Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.affiliation: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, 14424, Potsdam, Germany. daniel.kolarich@mpikg.mpg.de.
authors.forename: Daniel
authors.identifier: http://orcid.org/0000-0002-8452-1350
authors.initials: D
authors.lastname: Kolarich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D060748:Fetuins / Q000737:chemistry*; D006020:Glycopeptides / Q000032:analysis*; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D060748:Fetuins; D006020:Glycopeptides; D007136:Immunoglobulins
keywords: Collision energy stepping CID; Glycopeptide; Glycoproteomics; Immunoglobulin; N-glycan; O-glycan; Q-TOF; Synthetic glycopeptides