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dc.contributor.authorHinneburg, Hannes
dc.contributor.authorStavenhagen, Kathrin
dc.contributor.authorSchweiger-Hufnagel, Ulrike
dc.contributor.authorPengelley, Stuart
dc.contributor.authorJabs, Wolfgang
dc.contributor.authorSeeberger, Peter H
dc.contributor.authorSilva, Daniel Varon
dc.contributor.authorWuhrer, Manfred
dc.contributor.authorKolarich, Daniel
dc.date.accessioned2017-05-29T12:34:44Z
dc.date.available2017-05-29T12:34:44Z
dc.date.issued2016
dc.identifier.issn1044-0305
dc.identifier.doi10.1007/s13361-015-1308-6
dc.identifier.urihttp://hdl.handle.net/10072/337602
dc.description.abstractIn-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.
dc.description.peerreviewedYes
dc.languageEnglish
dc.language.isoeng
dc.publisherElsevier
dc.relation.ispartofpagefrom507
dc.relation.ispartofpageto519
dc.relation.ispartofissue3
dc.relation.ispartofjournalJournal of the American Society for Mass Spectrometry
dc.relation.ispartofvolume27
dc.subject.fieldofresearchAnalytical Chemistry not elsewhere classified
dc.subject.fieldofresearchAnalytical Chemistry
dc.subject.fieldofresearchMedicinal and Biomolecular Chemistry
dc.subject.fieldofresearchPhysical Chemistry (incl. Structural)
dc.subject.fieldofresearchcode030199
dc.subject.fieldofresearchcode0301
dc.subject.fieldofresearchcode0304
dc.subject.fieldofresearchcode0306
dc.titleThe Art of Destruction: Optimizing Collision Energies in Quadrupole-Time of Flight (Q-TOF) Instruments for Glycopeptide-Based Glycoproteomics
dc.typeJournal article
dc.type.descriptionC1 - Articles
dc.type.codeC - Journal Articles
dcterms.licensehttp://creativecommons.org/licenses/by/4.0/
dc.description.versionVersion of Record (VoR)
gro.rights.copyright© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
gro.hasfulltextFull Text
gro.griffith.authorKolarich, Daniel


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