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1.
Anal Chem ; 89(6): 3672-3680, 2017 03 21.
Article in English | MEDLINE | ID: mdl-28218834

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.


Subject(s)
Phosphopeptides/chemical synthesis , Serine/analysis , Threonine/analysis , Chromatography, Liquid , Electron Transport , Phosphopeptides/chemistry , Phosphorylation , Tandem Mass Spectrometry
2.
ACS Chem Biol ; 11(4): 1066-73, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26760216

ABSTRACT

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.


Subject(s)
Peptides/chemistry , Phosphoserine/chemistry , Proteins/chemistry
3.
Chembiochem ; 16(3): 415-23, 2015 Feb 09.
Article in English | MEDLINE | ID: mdl-25639821

ABSTRACT

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.


Subject(s)
Phosphopeptides/chemistry , Phosphopeptides/metabolism , Amino Acid Sequence , Diphosphates/metabolism , HeLa Cells/metabolism , Humans , Hydrolysis , Molecular Sequence Data , Phosphopeptides/chemical synthesis , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Engineering/methods , Protein Processing, Post-Translational , Protein Stability , Saccharomyces cerevisiae/metabolism
4.
J Am Chem Soc ; 136(1): 108-11, 2014 Jan 08.
Article in English | MEDLINE | ID: mdl-24350643

ABSTRACT

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.


Subject(s)
Inositol Phosphates/chemistry , Peptides/metabolism , Models, Molecular , Molecular Structure , Peptides/chemistry , Phosphorylation
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