A peptide-centric local stability assay enables proteome-scale identification of the protein targets and binding regions of diverse ligands.

By using a limited-proteolysis strategy that employs a large amount of trypsin to generate peptides directly from native proteins, we found that ligand-induced protein local stability shifts can be sensitively detected on a proteome-wide scale. This enabled us to develop the peptide-centric local stability assay, a modification-free approach that achieves unprecedented sensitivity in proteome-wide target identification and binding-region determination. We demonstrate the broad applications of the peptide-centric local stability assay by investigating interactions across various biological contexts.

Integrated Protein Solubility Shift Assays for Comprehensive Drug Target Identification on a Proteome-Wide Scale.

Target proteins are often stabilized after binding with a ligand and thereby typically become more resistant to denaturation. Based on this phenomenon, several methods without the need to covalently modify the ligand have been developed to identify target proteins for a specific ligand. These methods usually employ complicated workflows with high cost and limited throughput.

Highly effective identification of drug targets at the proteome level by pH-dependent protein precipitation.

Fully understanding the target spaces of drugs is essential for investigating the mechanism of drug action and side effects, as well as for drug discovery and repurposing. In this study, we present an energetics-based approach, termed pH-dependent protein precipitation (pHDPP), to probe the ligand-induced protein stability shift for proteome-wide drug target identification. We demonstrate that pHDPP works for a diverse array of ligands, including a folate derivative, an ATP analog, a CDK inhibitor and an immunosuppressant, enabling highly specific identification of target proteins from total cell lysates.

Matrix Thermal Shift Assay for Fast Construction of Multidimensional Ligand-Target Space.

Existing thermal shift-based mass spectrometry approaches are able to identify target proteins without chemical modification of the ligand, but they are suffering from complicated workflows with limited throughput. Herein, we present a new thermal shift-based method, termed matrix thermal shift assay (mTSA), for fast deconvolution of ligand-binding targets and binding affinities at the proteome level. In mTSA, a sample matrix, treated horizontally with five different compound concentrations and vertically with five technical replicates of each condition, was denatured at a single temperature to induce protein precipitation, and then, data-independent acquisition was employed for quick protein quantification.

Proteome-Wide Deconvolution of Drug Targets and Binding Sites by Lysine Reactivity Profiling.

Recently, numerous efforts have been devoted to identifying drug targets and binding sites in complex proteomes, which is of great importance in modern drug discovery. In this study, we developed a robust lysine reactivity profiling method to systematically study drug-binding targets and binding sites at the proteome level. This method is based on the principle that binding of a drug to a specific region of target proteins will change the reactivity of lysine residues that are located at this region, and these changes can be detected with an enrichable and lysine reactive probe.

Precipitate-Supported Thermal Proteome Profiling Coupled with Deep Learning for Comprehensive Screening of Drug Target Proteins.

Although thermal proteome profiling (TPP) acts as a popular modification-free approach for drug target deconvolution, some key problems are still limiting screening sensitivity. In the prevailing TPP workflow, only the soluble fractions are analyzed after thermal treatment, while the precipitate fractions that also contain abundant information of drug-induced stability shifts are discarded; the sigmoid melting curve fitting strategy used for data processing suffers from discriminations for a part of human proteome with multiple transitions. In this study, a precipitate-supported TPP (PSTPP) assay was presented for unbiased and comprehensive analysis of protein-drug interactions at the proteome level.

Mechanical stress induced protein precipitation method for drug target screening.

The process of protein precipitation can be used to decipher the interaction of ligand and protein. For example, the classic Thermal Proteome Profiling (TPP) method uses heating as the driving force for protein precipitation, to discover the drug target protein. Under heating or other denature forces, the target protein that binds with the drug compound will be more resistant to precipitation than the free protein.

Microparticle-Assisted Precipitation Screening Method for Robust Drug Target Identification.

While thermal proteome profiling (TPP) shines in the field of drug target screening by analyzing the soluble fraction of the proteome samples treated at high temperature, the counterpart, the insoluble precipitate, has been overlooked for a long time. The analysis of the precipitate is hampered by the inefficient sample processing procedure. Herein, we propose a novel method, termed microparticle-assisted precipitation screening (MAPS), for drug target identification.

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

Thermal proteome profiling is a powerful energetic-based chemical proteomics method to reveal the ligand-protein interaction. However, the costly multiplexed isotopic labeling reagent, mainly Multiplexed isobaric tandem mass tag (TMT), and the long mass spectrometric time limits the wide application of this method. Here a simple and cost-effective strategy by using dimethyl labeling technique instead of TMT labeling is reported to quantify proteins and by using the peptides derived from the same protein to determine significantly changed proteins in one LC-MS run.

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs.