Global Ion Suppression Limits the Potential of Mass Spectrometry Based Phosphoproteomics

Mass spectrometry
based proteomics has become the method of choice
for pinpointing and monitoring thousands of post-translational modifications,
predominately phosphorylation sites, in cellular signaling studies.
Critical for achieving this analytical depth is the enrichment of
phosphorylated peptides prior to liquid chromatography–mass
spectrometry (MS) analysis. Despite the high prevalence of this modification,
the numbers of identified phosphopeptides lag behind those achieved
for unmodified peptides, and the cause for this still remains controversial.
Here, we use an effective phosphatase protocol that considerably improves
global ionization efficiency and, therefore, the 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. In combination
with sensitive, label-free targeted MS for phosphorylation site validation,
our approach is ideally suited to exploring cellular phosphorylation
based signaling networks in high detail.