Enrichment Workflow for Pyrophosphoproteome Analysis.

The proteome-wide detection of different posttranslational modifications (PTMs) still poses a notable analytical challenge. Nevertheless, the identification of protein pyrophosphorylation sites using mass spectrometry (MS) was reported recently. The implementation of an enrichment workflow proved to be key to characterize this substoichiometric modification within complex cell lysate samples. Analysis of the enriched samples using data-dependent neutral-loss triggered electron transfer/higher-energy collision dissociation (DDNL EThcD) MS enabled the detection of endogenous protein pyrophosphorylation. In this chapter, the bottom-up pyrophosphoproteomics analysis, including an enrichment workflow, to identify protein pyrophosphorylation sites is described.