PhosphoProtein-Peptide Turnover Profiling, exploratory

We combine pulsed stable isotope labeling of amino acids (pSILAC) with enrichment of phosphorylated peptides in a method we term PhosphoProtein-Peptide Turnover Profiling (PhosphoPPToP). We apply 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.