Global ion suppression limits the potential of mass spectrometry based phosphoproteomics

Mass spectrometry-based proteomics has become the method of choice to pinpoint and monitor thousands of post-translational modifications, predominately phosphorylation sites, in cellular signaling studies. Critical for achieving this analytical depth is the enrichment for phosphorylated peptides prior to LC-MS analysis. Despite the high prevalence of this modification, the numbers of identified phosphopeptides are lower than those achieved for unmodified peptides, and the cause for this still remains controversial. Here we introduce an effective phosphatase protocol that considerably improves global ionization efficiency and therefore overall sensitivity and coverage of standard phosphoproteomics studies. We demonstrate the power of our method on the model system of Salmonella-infected macrophages by extending the current quantitative picture of immune signaling pathways involved in infection.