Sequential denaturation and protein precipitation assay (SDPP) improves the sensitivity for ligand target identification at the proteome level

Abstract

Background

Proteins can be denatured and precipitated under different denaturation conditions, and ligand bound proteins are more resistant to denaturing induced precipitation. Consequently, ligand target proteins can be identified by measuring solubility shifts. Nowadays, numerous methods have been developed to screen ligand target proteins based on different denaturation mechanisms. However, due to the different responses of proteins to different denaturation conditions, there is a significant complementary between these methods. Direct pooling of the denatured supernatants from different denaturation strategies can utilize this complementary, however, the pooling procedure inevitably averages the ligand-induced solubility shift across conditions, significantly compromising target identification sensitivity.

Results

To avoid pooling-induced signal compression and enhance identification sensitivity, we developed a novel solubility shift-based approach, termed Sequential Denaturation and Protein Precipitation assay (SDPP). In this strategy, multi-step sequential denaturation of individual sample was conducted, which greatly improves the identification sensitivity. We evaluated the performance of this method by screening the target proteins of Staurosporine, SDPP with thermal treatment followed by organic solvent treatment (TEMP-SL) performs better than other sequential denaturation and one-step denaturation approach. The number of kinase targets identified by SDPP (TEMP-SL) increased by 54 %, 38 %, 48 % and 21 %, respectively compared with the TEMP, SL, and pH experiments and IPSSA. We further applied this approach to identify the target proteins of endogenous metabolite cAMP. Except for revealing the known cAMP-PRKAR1A/PRKAR1B interaction, we also observed that the solubility shift of the C2orf88 responds to the cAMP binding.

Significance

This method conducts sequential denaturation on individual samples to induce stepwise protein precipitation, preserving and progressively accumulating the solubility shifts generated at each denaturation step. This cumulative effect amplifies the overall detectable solubility shift signal and greatly improves identification sensitivity, demonstrating great potential as an efficient and high-throughput strategy for proteome-wide ligand target identification.