Protein pyrophosphorylation is a minimally characterized post-translational modification (PTM) that has been implicated in several diseased states, most notably diabetes and obesity. To date, only in vitro evidence of this modification has been found using radiolabeling and biochemical methods. The main challenge to understanding the physiological significance of this PTM is that no methods exist for detecting this modification in cell lysates. Therefore, we are developing a chemical methodology to tag in vivo pyrophosphorylated proteins from a complex cellular environment. As a first necessary step, we have generated a robust methodology to chemically pyrophosphorylate peptides. This methodology is high-yielding, efficient in polar, protic solvents, and tolerant of functionally diverse peptide substrates. These results suggest a higher degree of phosphoserine nucleophilicity than previously anticipated and open the door for a wide range of applications. These pyrophosphopeptides have been used to understand the stability of this modification, as synthetic standards for investigating the biochemical pyrophosphosphorylation of peptides, and to optimize our chemical methodology to tag in vivo pyrophosphorylated proteins for mass spectrometry identification. This chemical derivatization methodology, and the peptide tools developed along the way, will allow us to gain a better appreciation for the regulatory roles of protein pyrophosphorylation.

Investigating protein pyrophosphorylation with chemically synthesized pyrophosphopeptides

247th National Meeting (2014)

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