Unambiguous Identification of Serine and Threonine Pyrophosphorylation Using Neutral-Loss-Triggered Electron-Transfer/Higher-Energy Collision Dissociation

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 method...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2017-03, Vol.89 (6), p.3672-3680
Main Authors: Penkert, Martin, Yates, Lisa M, Schùˆmann, Michael, Perlman, David, Fiedler, Dorothea, Krause, Eberhard
Format: Article
Language:English
Subjects:
Activation
Chromatography, Liquid
Electron Transport
Electrons
Energy
Energy of dissociation
Fragmentation
Genetics
Mass spectrometry
Peptides
Phosphopeptides - chemical synthesis
Phosphopeptides - chemistry
Phosphorylation
Precursors
Proteins
Serine - analysis
Spectra
Strategy
Tandem Mass Spectrometry
Threonine - analysis
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.6b05095