Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

Thermal proteome profiling (TPP) provides a powerfulapproach tostudying proteome-wide interactions of small therapeutic moleculesand their target and off-target proteins, complementing phenotypic-baseddrug screens. Detecting differences in thermal stability due to targetengagement requires high quantitative accuracy and consistent detection.Isobaric tandem mass tags (TMTs) are used to multiplex samples andincrease quantification precision in TPP analysis by data-dependentacquisition (DDA). However, advances in data-independent acquisition(DIA) can provide higher sensitivity and protein coverage with reducedcosts and sample preparation steps. Herein, we explored the performanceof different DIA-based label-free quantification approaches comparedto TMT-DDA for thermal shift quantitation. Acute myeloid leukemiacells were treated with losmapimod, a known inhibitor of MAPK14 (p38 & alpha;).Label-free DIA approaches, and particularly the library-free modein DIA-NN, were comparable of TMT-DDA in their ability to detect targetengagement of losmapimod with MAPK14 and one of its downstream targets,MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effectivealternative to labeled quantitation in the TPP pipeline.