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.

质谱法（Mass spectrometry）极大推动了复杂生物系统中磷酸化（phosphorylation）事件的规模化分析。尽管该领域已取得长足进展，但磷酸化位点的准确鉴定、定量分析及其生理相关性阐释，仍是极具挑战的课题。人类蛋白质组组织（HUPO）人类蛋白质组计划（HPP）的质谱资源工作组发起了磷酸肽挑战赛（Phosphopeptide Challenge），旨在为学界提供评估分析方法、学习实验流程与数据分析范式的共享资源，并通过对比标准数据集的分析结果，帮助研究人员搭建自有分析工作流：该标准数据集包含94种磷酸肽（phosphopeptides，涵盖丝氨酸（serine）、苏氨酸（threonine）与酪氨酸（tyrosine）磷酸化肽段）及其非磷酸化对应肽段，以纯样品与酵母背景基质两种形式制备，且不同肽段间设置了差异化比例梯度。参赛团队采用自选分析方法对两类样品进行检测，需完成以下任务：报告这些肽段的鉴定结果与位点定位信息，测定其相对丰度，并在酵母背景基质样品中完成磷酸肽的富集。本文梳理了22个实验室采用不同分析方法、仪器与数据分析软件所获得的实验结果，并采用统一的软件分析管线对提交的原始数据进行了重新解析，重点阐明了磷酸化位点准确鉴定过程中的优势与现存挑战。本协作研究产生的全部数据均作为共享资源发布，以期推动更适配各类磷酸蛋白质组学应用场景的方法与工具的开发。所有提交的原始数据与搜索结果均已上传至MassIVE数据库（https://massive.ucsd.edu/），对应数据集编号为MSV000085932，ProteomeXchange标识符为PXD020801。