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.