Experimental and data analysis advances in thermal proteome profiling

Abstract

Method development for mass spectrometry (MS)-based thermal shift proteomic assays have advanced to probe small molecules with known and unknown protein-ligand interaction mechanisms and specificity, which is predominantly used in characterization of drug-protein interactions. In the discovery of target and off-target protein-ligand interactions, a thorough investigation of method development and their impact on the sensitivity and accuracy of protein-small molecule and protein-protein interactions is warranted. In this review, we discuss areas of improvement at each stage of thermal proteome profiling data analysis that includes processing of MS-based data, method development, and their effect on the overall quality of thermal proteome profiles. We also overview the optimization of experimental strategies and prioritization of an increased number of independent biological replicates over the number of evaluated temperatures.

Keywords: CP: biotechnology; cellular thermal shift assays; drug discovery; protein-ligand interactions; target engagement; thermal proteomic profiling.

Figure 1

Schematic representation of workflows (A) General experimental workflow for one-dimensional thermal proteome profiling and (B) general experimental workflow for two-dimensional thermal proteome profiling.

Figure 2

Challenges for thermal shift assays for each of the replicates acquired for the same sample type (i.e., either treated or control) (A) The implementation of carrier proteomes into adjacent channels; (B) the presence of missing values; (C) aggressive filtering due to missing values and channel interference; and (D) overfitting the data with more flexible models. Colors indicate separate temperature challenges; circles and triangles represent two biological replicates.