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.

Pyrophosphorylation via selective phosphoprotein derivatization

“…Peptides were fragmented by electron-transfer/higher-energy dissociation (EThcD) to allow for reliable localization of different types of phosphorylation while enabling high sequence coverage. 7 , 22 , 37 The analysis confirmed the pyrophosphorylation of myoglobin at S127 ( Fig. 6a ) and over 90% conversion of the starting monophosphorylated protein.…”

“…1b ). 20 , 21 While the peptides facilitated the development of analytical tools, 22 , 23 a long-term goal is to demonstrate the effect of pyrophosphorylation on protein structure and function. The ability to produce site-specifically and stoichiometrically pyrophosphorylated proteins would provide the opportunity for full biochemical and biophysical characterization of this modification.…”

The Role of Electron Transfer Dissociation in Modern Proteomics

“… 127 Neutral loss-triggered decision trees have also been employed for analysis of pyro-phosphorylation, a more rare version of serine and threonine phosphorylation, where EThcD was used to confidently localize pyro-phosphorylation sites and rule out possibilities of multiple standard (e.g., “non-pyro”) phosphosites. 128 …”

Identity and functions of inorganic and inositol polyphosphates in plants

“…Protein pyrophosphorylation has the potential to affect the function of target proteins and directly transduce PP-InsP metabolism into a signaling output; however, it has not yet been possible to directly identify pyrophosphorylated proteins from cell extracts. Advances in mass spectrometry methods (Penkert et al, 2017) are likely overcome this obstacle soon. Functional studies of non-enzymatic processes are inherently problematic, but the fact that PP-InsP-mediated pyrophosphorylation requires pre-phosphorylation of the serine residue (typically by casein kinase 2 and possibly other kinases) further hinders its in vivo characterization, since mutating the substrate serine would prevent the distinction of the kinasemediated phosphorylation and polyphosphorylation.…”

“…Without the possibility of genetically dissecting this process, one has to rely on in vitro biochemical methods; nevertheless, some sort of in vivo validation will be required to fully characterize pyrophosphorylation as a signaling mechanism. A promising new mass spectrometry technique was able to distinguish peptide pyrophosphorylation in vitro (Penkert et al, 2017) and may finally allow its detection from cell lysates. In addition, a fluorescent sensor designed to specifically bind diphosphate esters may provide agility in the identification and relative-quantification of protein pyrophosphorylation (Williams & Fiedler, 2015).…”