PhosphoProtein-Peptide Turnover Profiling, validation

We combined pulsed stable isotope labeling of amino acids (pSILAC) with enrichment of phosphorylated peptides in a method we term PhosphoProtein-Peptide Turnover Profiling (PhosphoPPToP). We applied the method to HeLa cells to find peptides whose clearance rates differ from the protein median. Using theoretical modelling, we show that readouts from PPToP are primarily defined by the wiring of the PTM-modification network and not by effects of the PTM on a protein's proteolytic stability. Thus PPToP can be used to identify PTMs occurring, e.g., preferentially early or late in a protein's lifetime. This enables identification of, e.g., protein maturation or protein complex assembly intermediates. This dataset covers experimental testing of identified phosphosites with PPToP. Here, proteins of interest are expressed as GFP-fusion constructs in HeLa cells and subjected to a SILAC pulse. Thereafter, proteins are pulled-down with anti-GFP beads to isolate the exogenously expressed constructs. We multiplex SILAC timepoints from wild-type and mutant constructs for every given protein of interest using TMT16.