Thesaurus: quantifying phosphoprotein positional isomers

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 new positional isomers using site-specific fragment ions 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 quantitative profiles, indicating regulation by different kinases.