Purpose
Cellular thermal shift assay (CETSA), or thermal proteome profiling (TPP), is the method of choice for characterizing protein (off)targets and mechanism of action of small molecule drugs inside living cell. In this study we show further method advancements using the TMTpro TM 32plex reagent with real time search SPS-MS3 to further improve both throughput and quality of thermal proteome profiling for characterizing protein targets of natural product Alantolactone.
Methods
Human myelogenous leukemia cells (K562), and cell lysate were treated with Alantolactone for TPP/CETSA analysis. Thermally denatured proteins were removed using centrifugation, and remaining soluble proteins were digested with trypsin and labeled with TMTpro 32plex. For protein integral solubility assay experiments (PISA), temperature points were pooling prior to labelling. Samples were fractionated using high-pH chromatography and analyzed with a Thermo Scientific TM Vanquish TM Neo UHPLC and Thermo Scientific TM Orbitrap TM Ascend TM MultiOmics Tribrid TM mass spectrometer and data was processed with Proteome Discoverer 3.2.
Results
Using 32plex enabled experiments of compound-induced thermal stability changes at four different concentrations, in intact and lysed cells in a single sample set. This allowed us to identify 12 more potential ALT targets and distinguish between thermal stability changes caused by direct binding and those induced by downstream signaling.
In the second experiment, four eight-point melt curves could be combined within one multiplexed sample. Compound-induced changes in protein abundance, solubility, stability could be visualized and deconvoluted. While proteins like ARK1A1 and PTGR2 appear as targets of ALT, different effects on ARK1B1, AKR1C2 and ARK1C3 were observed.
PISA results acquired with significantly decreased analysis time through reduced fraction numbers and shorter gradients are compared.
Conclusions
While proteins like ARK1A1 and PTGR2 appear as targets of ALT, with changes only in protein thermal stability, different effects on ARK1B1, AKR1C2 and ARK1C3 were observed.