蛋白质 z-bio 2025-02-12 402

　　Identification of ligand target proteins and binding sites, determination of local affinity

　　Supported by the National Natural Science Foundation of China (Grant No. 22204033, 92153302, 22137002), Ye Mingliang team of Dalian Institute of Chemical Physics, Chinese Academy of Sciences cooperated with Luo Cheng team of Institute of Materia Medica, Chinese Academy of Sciences, and made important progress in the field of new methods for proteomic identification of ligand target proteins. Related achievements are based on "A peptide centric local stability assay enables proteome scale identification of the protein targets and binding regions of diverse ligands and), published in Nature Methods recently. Paper link https://www.nature.com/articles/s41592-024-02553-7 .

　　There are various ligand molecules in the intracellular and extracellular environment, including metabolites, metal ions, nucleic acids, proteins, drugs, etc. Their interactions with proteins affect the physiological and pathological processes of organisms. Identifying the binding proteins and binding sites of ligands can help reveal the mechanism of action of ligands in life activities, which is of great significance for understanding complex life systems, deciphering disease mechanisms, and promoting drug development. The traditional ligand target proteomics identification method requires designing and optimizing probe synthesis schemes that maintain ligand activity for different ligands, which not only lacks broad-spectrum applicability but also makes it difficult to identify ligand target proteins with weak interactions.

　　In response to these issues, the aforementioned team has developed a peptide centered protein local stability assay (PELSA). This method does not require chemical modification of ligands and does not depend on affinity size. It can directly discover proteins that bind to ligands and undergo local stability changes in complex systems such as cell lysate, thereby achieving systematic analysis of ligand binding proteins, binding sites, and local affinity. The identification sensitivity of this method for the target protein of the model drug cyclosporine has increased by 12 times and 2.4 times compared to existing similar technologies such as LiP-MS and TPP, respectively. In addition, the dose-dependent PELSA method can determine local affinity, thereby revealing the dynamic changes in the three-dimensional structure of proteins after binding to ligands under physiological conditions. The team applied the PELSA method to identify binding proteins of various ligands such as drugs, metal ions, post-translational modified peptides, and antibodies, all of which demonstrated highly sensitive target protein identification performance and accurate binding region localization ability, proving that the PELSA method can be used as a universal analysis platform without chemical modification of ligands, and is widely applicable to target protein analysis of different structural ligands.