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 <i>in silico</i> predicted spectral libraries and thus rapid and sensitive analysis of DIA phosphoproteomics experiments.