Quantitative profiling of PTM stoichiometry by DNA mass tags

Protein post-translational modification (PTM) serves as an important mechanism for regulating protein function. Accurate assay of PTM stoichiometry, or PTM occupancy, which refers to the proportion of proteins that contain specific modifications, is important for understanding the function of PTMs. We previously developed a novel chemoproteomic strategy "STO-MS" to quantify the PTM stoichiometry in complex biological samples, which employs a resolvable polymer mass tag to differentiate modified proteins and utilizes liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques to measure PTM stoichiometry. However, the resolution of STO-MS is constrained by the relatively low molecular weight of the mass tag, and the incorporation of isotopic labels not only complicates the sample preparation but also restricts the measurement throughput. To address these challenges, we herein developed "STO-MS+", an enhanced workflow, that incorporates an optimized DNA mass tag and employs a label-free quantitative data analysis approach. We applied STO-MS+ to measure stoichiometry of three distinct PTMs, including endogenous carbonylation induced by arachidonic acid (AA), itaconation, and endogenous O-GlcNAcylation. Our work marks a notable improvement in chemoproteomic methodologies for quantifying post-translational modifications and provides a powerful analytical tool for PTM research.