The Introduction of Detergents in Thermal Proteome Profiling Requires Lowering the Applied Temperatures for Efficient Target Protein Identification

Abstract

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification.

Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling

“…Omitting the labeling step allows one to follow changes in protein expression levels while retaining information about protein stability. Hence, with label-free quantitation, more information is obtained from a single sample set and allows the user to create a multilayered information data set, previously shown for TPP workflows. − …”