TIPP: Un nuevo enfoque de precipitación de proteínas inducida por reacción con iodoacetamida para la identificación...

Ligand-bound proteins have higher stability and thereby greater resistance to denaturing-induced precipitation than free proteins, which forms the basis of several modification-free ligand target protein identification methods. However, due to the vast sequence diversity of cellular proteins, different proteins respond differently to distinct denaturation mechanisms, and a single denaturation mechanism can only cover certain target proteins. Therefore, developing efficient target protein identification methods based on novel denaturation mechanisms is of great significance for improving the coverage of target identification. Herein, we propose a novel target protein identification method relying on a new precipitation mechanism, namely, Target Identification by Iodoacetamide reaction-induced Protein Precipitation (TIPP). The TIPP method uses iodoacetamide (IAA), a thiol-blocking reagent commonly used in shotgun proteomics, to irreversibly alkylate the thiol groups of free cysteine residues, thereby inducing protein denaturation and precipitation. Under IAA treatment, proteins in different conformational states have different exposures of free thiol groups, resulting in different precipitation efficiencies, which is the key theoretical basis for the design of the TIPP method for target protein identifications. The feasibility of the developed TIPP method was evaluated by identifying target proteins of various ligand compounds, including MTX, TG101348, and staurosporine. In addition, this method was also used to monitor the target proteins of metal ions with the metal ion chelator TPEN. The developed method has similar efficiency and good complementarity with the traditional method, and can be combined with the traditional method to increase the coverage of target identification. Therefore, TIPP is expected to become a powerful strategy to achieve comprehensive ligand-target identification by complementing other drug target identification methods.