In this study,we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples,such as purified protein complexes.This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM).Specifically,low confident phosphopeptides identified from the data dependent acquisition (DDA) dataset were used to build a pseudo-targeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence.