16-plex isobaric mass tags enable streamlined proteomics-based drug mechanism-of-action studies

ABSTRACT: Multiplexed quantitative proteomics enables the development of complex workflows for studying the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity with which such experiments could be performed. Here we evaluate a new generation of 16-plex isobaric mass tags and demonstrate similar sensitivity and accuracy of quantification as the previously described TMT reagents. The TMT16 tags enabled the sensitive and time efficient identification of staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over 7 concentrations per temperature enabled comprehensive selectivity profiling of staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

SUBMITTER: Toby Mathieson  

LAB HEAD: Marcus Bantscheff

PROVIDER: PXD020848 | Pride | 2021-02-25

REPOSITORIES: Pride