TY - JOUR T1 - Diverse Hotspot Thermal Profiling Methods Detect Phosphorylation-Dependent Changes in Protein Stability JF - bioRxiv DO - 10.1101/2021.05.01.441686 SP - 2021.05.01.441686 AU - Benjamin D. Stein AU - Jun X. Huang AU - David Wu AU - Lewis C. Cantley AU - Raymond E. Moellering Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/05/01/2021.05.01.441686.abstract N2 - Hotspot thermal profiling (HTP) methods utilize modified-peptide level information in order to interrogate proteoform-specific stability inside of live cells. The first demonstration of HTP involved the integration of phosphopeptide enrichment into a TMT-based, single-LC separation thermal profiling workflow1. Here we present a new ‘label-fractionate-enrich’ (LFE)-HTP method that involves high-pH reverse phase fractionation of TMT-labeled peptides prior to phosphopeptide enrichment, followed by peptide detection and quantitation using multi-notch LC-MS3. We find that LFE-HTP, while more resource intensive, improves the depth and precision of (phospho)proteoform coverage relative to the initial published HTP workflow. The fraction of detected phosphorylation sites that are significantly perturbed in this new dataset are consistent with those seen in our previous study, as well as those published by others, when compared head-to-head with the same analysis pipelines. Likewise, many ‘hotspot’ phosphorylation sites identified in our paper are consistently reproduced by LFE-HTP as well as other modified HTP methods. The LFE-HTP dataset contains many novel ‘hotspot’ phosphorylation sites that regulate the stability of diverse proteins, including phosphosites in the central glycolytic enzyme Aldolase A that are associated with monomer-to-oligomer formation, enzymatic activity and metabolic regulation in cancer cells. Our comparative analyses confirm that several variants of the HTP method can track modified proteoforms in live cells to detect and prioritize PTM-dependent changes in protein stability that may be associated with function.Competing Interest StatementThe authors have declared no competing interest. ER -