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 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.

A Stable Pyrophosphoserine Analog for Incorporation into Peptides and Proteins.

Protein pyrophosphorylation is a covalent modification of proteins, mediated by the inositol pyrophosphate messengers. Although the inositol pyrophosphates have been linked to a range of cellular processes, the role of protein pyrophosphorylation remains minimally characterized in vivo. The inherent instability of the phosphoanhydride bond has hampered the development of useful bioanalytical techniques to interrogate this novel signaling mechanism. Here, we describe the preparation of a pyrophosphoserine analog containing a stable methylene-bisphosphonate group that is compatible with solid-phase peptide synthesis. The resulting peptides demonstrate enhanced stability in Eukaryotic cell lysates and mammalian plasma and display resistance toward chemical degradation, when compared to the corresponding pyrophosphopeptides. In addition, the peptides containing the stable pyrophosphoserine analog are highly compatible with common ligation methods, such as native chemical ligation, maleimide conjugation, and glutaraldehyde ligation. The bisphosphonate-containing peptides will, therefore, be well-suited for future pyrophosphoserine antibody generation and affinity capture of pyrophosphoprotein binding partners and provide a key entry point to study the regulatory role of protein pyrophosphorylation.

Establishing the stability and reversibility of protein pyrophosphorylation with synthetic peptides.

Protein pyrophosphorylation is emerging as a new post-translational modification, yet its role in cellular signaling remains poorly characterized. Important factors in determining the biological relevance of pyrophosphorylation include understanding the chemical and biochemical stability of the pyrophosphoryl group and elucidating the reversibility of modification in a cellular context. Towards this end, we prepared a series of synthetic pyrophosphopeptides, which were utilized to demonstrate that the modification is quite inert over a wide pH range but can be removed biochemically by alkaline phosphatases. Importantly, we observed enzyme-dependent removal of the pyrophosphate in mammalian and yeast cell lysates using the synthetic pyrophosphopeptides. The findings provide evidence for the reversibility of pyrophosphorylation and thereby highlight the potential impact of this modification on cellular signal transduction pathways.

Chemical pyrophosphorylation of functionally diverse peptides.

A highly selective and convenient method for the synthesis of pyrophosphopeptides in solution is reported. The remarkable compatibility with functional groups (alcohol, thiol, amine, carboxylic acid) in the peptide substrates suggests that the intrinsic nucleophilicity of the phosphoserine residue is much higher than previously appreciated. Because the methodology operates in polar solvents, including water, a broad range of pyrophosphopeptides can be accessed. We envision these peptides will find widespread applications in the development of mass spectrometry and antibody-based detection methods for pyrophosphoproteins.