Stability-based approaches in chemoproteomics

Abstract

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Keywords: chemical proteomics; chemoproteomics; drug discovery; limited proteolysis; mass spectrometry; proteome integral solubility alteration; target deconvolution; target engagement; thermal proteome profiling.

Figure 1.

Schematic representation of chemoproteomic approaches based on thermal denaturation. All of these methods are technically applicable to cell lysates, live cells and tissues. (A) TPP-TR: samples are treated with a single dose of compound and subject to an increasing temperature gradient. Protein abundance in soluble fraction of each temperature labelled with TMT, pooled and fractionated. Melting curves constructed in the presence and absence of the drug are used to calculate shift in melting point (T_m). (B) TPP-CCR: cells are treated with different compound concentrations and a vehicle control at a fixed temperature, generating affinity data. (C) 2D-TPP: dose-dependent thermal stabilisation profiling. (D) Isothermal shift assay (iTSA) measures difference in the soluble protein fraction at a single temperature, requiring only two TMT channels per biological replicate. By freeing up available TMT channels, more replicates can be condensed into a single experiment as fractionation is still performed. This leads to a four-fold increase in throughput, allowing for more replicate analyses, which improves statistical power.

Figure 2.

Schematic of chemoproteomic approaches based on limited proteolytic degradation. All methods are currently applicable to cell lysates only. (A) DARTS identifies proteins with enhanced resistance to proteolysis, by enriching for target proteins by SDS–PAGE. Detectable targets will have higher band intensity compared to the vehicle-treated sample. Both regions are excised for in-gel digestion and MS analysis, providing a rapid approach for target identification. (B) Lysates are treated over a concentration range and then aliquot in half. One-half is subject to limited proteolysis under native conditions, then both aliquots are subject to complete digestion under denaturing conditions, analysed with DIA. Peptides with normalised dose-dependent changes in abundance are scored using LiP-Quant score, ranking the list of targets and providing site-specific information.

Figure 3.

Schematic of chemoproteomic approaches based on chemical denaturation or precipitation. (A) Stability of proteins from rates of oxidation (SPROX): Cell lysates are treated with increasing concentrations of chaotropic reagent to unfold proteins, exposing methionine residues for oxidation by addition of H₂O₂. (B) Solvent-induced protein precipitation (SIP): Lysates are treated with increasing concentrations of solvent, causing protein precipitation. Soluble fractions are subject to dimethyl labelling, and vehicle (heavy) and treatment (light) conditions combined per concentration for LC–MS analysis. Fold change between vehicle and treated conditions are compared. (C) Solvent proteome profiling (SPP) combines the principle of SIP with TMT quantification. Each concentration is labelled, combined, fractionated and analysed to construct denaturation curves in the presence and absence of the drug to calculate shift in the concentration of solvent at which 50% of the protein is unfolded.

Figure 4.

Schematic of chemoproteomic approaches measuring alterations in solubility, applicable to live cells and lysates. (A) Proteome integral solubility alteration – temperature (PISA-T): utilises a temperature-centric approach, pooling soluble fractions across temperature points prior to TMT labelling and fractionation, to measure protein abundance and difference in area under the melting curve. A 2D format is optional for added specificity. (B) PISA-I, measures protein solubility in live cells post-denaturation using a kosmotropic gradient. Similarly, soluble fractions are pooled prior to TMT labelling followed by fractionation. An advantage is that more TMT channels are available compared with TPP, allowing more samples to be compared quantitatively in a single experiment.