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

Thermal proteome profiling (TPP) is a powerful energetic-based chemical proteomics method to reveal the ligand-protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly TMT, and the long mass spectrometric time limits the wide application of this method. Here we report a simple and cost-effective strategy by using dimethyl labeling technique instead of TMT labeling 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 was validated by identifying the known targets of methotrexate (MTX) and geldanamycin. In addition, several potential off-targets involved in detoxification of reactive oxygen species (ROS) pathway were also discovered for geldanamycin. This method was further applied to map the interactome of ATP in the 293T cell lysate by using ATP analogue, AMP-PNP, as the ligand. As a result, a total of 123 AMP-PNP-sensitive proteins were found, of which 59 proteins were stabilized by AMP-PNP. Approximately 53% and 20% of these stabilized candidate protein targets were 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.