A Simplified Thermal Proteome Profiling Approach to Screen Protein Targets of a Ligand.

ABSTRACT

Thermal proteome profiling is a powerful energetic-based chemical proteomics method to reveal the ligand-protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost-effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC-MS run. This method is validated by identifying the known targets of methotrexate and geldanamycin. In addition, several potential off-targets involved in detoxification of reactive oxygen species pathway are also discovered for geldanamycin. This method is further applied to map the interactome of adenosine triphosphate (ATP) in the 293T cell lysate by using ATP analogue, adenylyl imidodiphosphate (AMP-PNP), as the ligand. As a result, a total of 123 AMP-PNP-sensitive proteins are found, of which 59 proteins are stabilized by AMP-PNP. Approximately 53% and 20% of these stabilized candidate protein targets are known as ATP and RNA binding proteins. Overall, above results demonstrated that this approach could be a valuable platform for the unbiased target proteins identification with reduced reagent cost and mass spectrometric time.