A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics

Abstract

Mass-spectrometry-based phosphoproteomics has become indispensable for understanding cellular signaling in complex biological systems. Despite the central role of protein phosphorylation, the field still lacks inexpensive, regenerable, and diverse phosphopeptides with ground-truth phosphorylation positions. Here, we present Iterative Synthetically Phosphorylated Isomers (iSPI), a proteome-scale library of human-derived phosphoserine-containing phosphopeptides that is inexpensive, regenerable, and diverse, with precisely known positions of phosphorylation. We demonstrate possible uses of iSPI, including use as a phosphopeptide standard, a tool to evaluate and optimize phosphorylation-site localization algorithms, and a benchmark to compare performance across data analysis pipelines. We also present AScorePro, an updated version of the AScore algorithm specifically optimized for phosphorylation-site localization in higher energy fragmentation spectra, and the FLR viewer, a web tool for phosphorylation-site localization, to enable community use of the iSPI resource. iSPI and its associated data constitute a useful, multi-purpose resource for the phosphoproteomics community.

Extended Data Fig. 1 |. Using the iSPI to compare phosphoproteomics pipelines.

The datasets for the three subpools were analyzed using four different pipelines. Each pipeline included a database searching algorithm and a site localization tool. a) Similar to Fig. 2, receiver operating curves are shown displaying the number of combined sites from the three pools on the y-axis as a function of the FLR on the x-axis. Vertical grey dotted lines represent empirical FLR’s of 0.01 and 0.05 respectively. Labelled points represent commonly used localization cutoffs: 13 for AScore and AScorePro, 0.75 localization probability for MaxQuant, and 0.95 site probability for proteome discoverer. At an empirical FLR of 0.05, differences are apparent. b) The number of phosphorylation sites per pool is shown for label free peptides at an empirical 5% FLR. c) The number of phosphorylation sites per pool is shown for TMTpro-labeled peptides at an empirical 5% FLR. Bar represents the mean. Error bars represent the standard error of the mean for n = 3 subpools.

Fig. 1 |. iSPI is a multi-purpose, regenerable, proteome-scale human phosphopeptide resource for phosphoproteoimcs.

a, iSPI generates authentically phosphorylated peptides derived from the human phosphoserine proteome that can be used as standards and for instrumental method optimization. The raw data files can be used to generate ground-truth phosphopeptide spectral libraries. The FLR determination tool enables researchers to investigate their own data and data processing workflows. AScorePro is an improved version of AScore, with improved phosphorylation-site localization. b, Workflow for determining FLR using iSPI. For this report, 3 library subpools of ~11,000 phosphorylated peptides were expressed using the Phosphoserine Orthogonal Translation System (pSerOTS), which, after purification, digestion, and enrichment, generated 3 pools of peptides with known phosphorylation positions. Note that, for clarity, each subpool never contained the same phosphopeptide phosphorylated on two sites (no positional isomers). Tryptic phosphorylated peptides were analyzed under various collision modes with and without TMTpro labeling, and search-engine outputs were compared with the known positions to determine FLR. LC–MS/MS, liquid chromatography–tandem mass spectrometry.

Fig. 2 |. Using iSPI to evaluate collision modes and labeling states, as well as to improve the AScore algorithm.

Receiver operating curves were determined; the y axis represents the total number of phosphorylation sites across the three subpools from Figure 1, and the x axis represents the precise FLR. Vertical gray dotted lines represent empirical FLRs of 0.01 and 0.05; horizontal lines represent 10,000 and 7,000 phosphorylation sites for label-free and TMTpro-labeled peptides, respectively. Labeled points represent discrete AScore cutoffs. Bold red numbers and arrows represent the differences in FLR (horizontal arrow) or identified sites (vertical arrow) between curves. For clarity, the combined totals of the three pools are plotted. a, Comparison of collected phosphopeptide spectra using CID, HCD, or MSA collision modes under label-free (left) and TMTpro-labeled (right) states. b, Comparison of AScore and AScorePro algorithms. AScorePro specifically improves FLR for data collected using HCD fragmentation.