Quantitative profiling of PTM stoichiometry by resolvable mass tags

Post-translational modifications (PTMs) play important roles in modulating the biological functions of proteins. Stoichiometry, which quantifies the modification percentage, is a critical factor for any given PTM. In this work, we developed a chemoproteomic strategy called “STO-MS” to systematically quantify the PTM stoichiometry in complex biological samples. This strategy employs a resolvable mass tag to differentiate proteoforms with different numbers of modifications and utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to measure PTM stoichiometry at the proteomic level. As a proof-of-concept, we successfully determined the stoichiometry of 197 proteins modified by 4-hydroxynonenal (HNE), a well-characterized lipid-derived electrophile and biomarker for oxidative stress. Our work expands the toolbox for quantification of PTM stoichiometry and sheds light on understanding the biological significance of PTMs in oxidative stress.