Pho-Tip: One-Pot Dephosphorylation for Rapid and Sensitive Analysis of DIA Phosphoproteomics Data

Recent advances in instrumentation and data processing
have transformed
data-independent acquisition (DIA) proteomics into a reliable technology
for quantitative profiling of post-translational modifications. However,
analysis of DIA phosphoproteomics data is challenging due to the large
search space, wherein all combinations of phosphosites on a peptide
need to be considered. Current approaches therefore face significant
hurdles in detecting low-abundant phosphorylated peptides, in particular
when working with low sample amounts. Here we introduce Pho-Tip, a
lossless one-pot dephosphorylation strategy. We show that Pho-Tip
enables comprehensive mapping of phosphorylated peptide sequences,
facilitating streamlined creation of experiment-focused in
silico predicted spectral libraries and thus rapid and sensitive
analysis of DIA phosphoproteomics experiments.