Insights from the First Phosphopeptide Challenge of the MS Resource Pillar of the HUPO Human Proteome Project

Mass spectrometry has greatly improved the analysis of phosphorylation events in complex biological systems and on a large scale. Despite considerable progress, the correct identification of phosphorylated sites, their quantification, and their interpretation regarding physiological relevance remain challenging. The MS Resource Pillar of the Human Proteome Organization (HUPO) Human Proteome Project (HPP) initiated the Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and data analysis routines, and establish their own workflows by comparing results obtained from a standard set of 94 phosphopeptides (serine, threonine, tyrosine) and their nonphosphorylated counterparts mixed at different ratios in a neat sample and a yeast background. Participants analyzed both samples with their method(s) of choice to report the identification and site localization of these peptides, determine their relative abundances, and enrich for the phosphorylated peptides in the yeast background. We discuss the results from 22 laboratories that used a range of different methods, instruments, and analysis software. We reanalyzed submitted data with a single software pipeline and highlight the successes and challenges in correct phosphosite localization. All of the data from this collaborative endeavor are shared as a resource to encourage the development of even better methods and tools for diverse phosphoproteomic applications. All submitted data and search results were uploaded to MassIVE (https://massive.ucsd.edu/) as data set MSV000085932 with ProteomeXchange identifier PXD020801.