Thesaurus: quantifying phosphopeptide positional isomers

Abstract

Proteins can be phosphorylated at neighboring sites resulting in different functional states, and studying the regulation of these sites has been challenging. Here we present Thesaurus, a search engine that detects and quantifies phosphopeptide positional isomers from parallel reaction monitoring and data independent acquisition mass spectrometry experiments. We apply Thesaurus to analyze phosphorylation events in the PI3K/AKT signaling pathway and show neighboring sites with distinct regulation.Hundreds of thousands of amino acids in thousands of proteins are estimated to be actively phosphorylated in every human cell (1). Many proteins are phosphorylated at neighboring sites (2) and over half of sites in multi-phosphorylated proteins are within four amino acids of each other (3). Several well-studied proteins make use of neighboring phosphorylation sites to act as switches (MAPK (4), CDC4 (5)), timers (PER ( 6)) or as negative inhibition toggles (IRS1 ( 7)) but global analysis of these phosphorylation clusters has remained impractical. Tandem mass spectrometry (MS/MS) of tryptic peptides is a key tool in discovering and quantifying sites of protein phosphorylation. Typical phosphoproteomic workflows use data dependent acquisition (DDA) to collect MS/MS spectra based on precursor m/z as peptides chromatographically elute. Site localization software tools such as Ascore (8) assign the most likely phosphorylation position for each peptide using sitespecific fragment ions. To increase the number of distinct peptides that are sampled, DDA Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

R2‐P2 rapid‐robotic phosphoproteomics enables multidimensional cell signaling studies

“…The MS measurements resulted in 82 injections with 60‐min peptide separation gradients; together with the sample preparation (starting from cell pellets), the experiment was completed in 8 days (Fig A). For the analysis of the acquired data, we used Skyline to generate the spectral library from the DDA runs (MacLean et al , ), EncylopeDIA to generate the chromatogram library from the narrow‐window DIA runs (Searle et al , ), and Thesaurus to identify, site‐localize, and quantify phosphopeptides in the wide‐window DIA runs (Searle et al , ).…”

Generating high quality libraries for DIA MS with empirically corrected peptide predictions

“…7a), can share fragment ions ( Supplementary Fig. 7b) and must be localized as if they contained post-translational modifications (PTMs) 29,30 if they fall in the same precursor isolation window.…”

Quantitative Method for Assessing the Role of Lysine & Arginine Post-Translational Modifications in Nonalcoholic Steatohepatitis

“…It has recently become possible to computationally assess the strength of PTM localization of peptidoforms which contain positional isomers using tools which score distinguishing fragment ions between isomers to differentiate and assess the correct site of modification. 7,9,10 However, these tools were all tested and optimized for phosphopeptide positional isomer localization using synthesized peptides and designed to be used on phosphopeptide enrichments; a significantly less complex sample matrix than complex cellular lysate. We chose to use Thesaurus 0.9.4 for modified peptidoform localizations, a graphical interface easily operated by an end-user which contains the ability to score and localize the Lys and Arg PTM's used in this study increasing the confidence of PTMs identified with our approach.…”

“…The added benefit of DIA based sampling to reduce missing data and thereby improve the consistency of peptide detection and quantitation promises the continued advancement for the MS-based study of low abundant PTMs. In addition, recently published algorithms PIQUed, 7 Peptide Collapse plugin for Perseus, 8 Inference of Peptidoforms (IPF), 9 and Thesaurus, 10 claim reduced false site localization and greater PTM recovery providing an alternative to the classical approach to elucidate PTM dynamics in complex biological systems and a more complete understanding of the complexity of cellular signaling, which we have harnessed. 4,5,9,[11][12][13] In proteins, methylation generally occurs on Arg and Lys residues with the ε-amino group of Lys being either mono, di, or trimethylated by protein lysine methyltransferases while Arg can be mono or dimethylated by protein arginine methyltransferases.…”