title: Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation.
abstract: Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Robert-Rössle Str. 10, 13125 Berlin, Germany.|Humboldt Universität zu Berlin , Department of Chemistry, Brook-Taylor-Straße 2, 12489 Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Penkert
authors.affiliation: Princeton University , Department of Chemistry, Frick Chemistry Building, Washington Road, Princeton, New Jersey 08544, United States.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Yates
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Robert-Rössle Str. 10, 13125 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schümann
authors.affiliation: Princeton University , Department of Molecular Biology, 119 Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, United States.
authors.forename: David
authors.initials: D
authors.lastname: Perlman
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Robert-Rössle Str. 10, 13125 Berlin, Germany.|Humboldt Universität zu Berlin , Department of Chemistry, Brook-Taylor-Straße 2, 12489 Berlin, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Fiedler
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Robert-Rössle Str. 10, 13125 Berlin, Germany.
authors.forename: Eberhard
authors.identifier: 0000-0001-5019-522X
authors.initials: E
authors.lastname: Krause
mesh_terms: D002853:Chromatography, Liquid; D004579:Electron Transport; D010748:Phosphopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D010766:Phosphorylation; D012694:Serine / Q000032:analysis*; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000032:analysis*
chemical_list: D010748:Phosphopeptides; D013912:Threonine; D012694:Serine