Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm 17177, Sweden.
Department of Pharmacological & Technological Chemistry, I.M. Sechenov First Moscow State Medical University, Moscow 119146, Russia.
Anal Chem. 2022 May 17;94(19):7066-7074. doi: 10.1021/acs.analchem.2c00391. Epub 2022 May 4.
Unbiased drug target engagement deconvolution and mechanism of action elucidation are major challenges in drug development. Modification-free target engagement methods, such as thermal proteome profiling, have gained increasing popularity in the last several years. However, these methods have limitations, and, in any case, new orthogonal approaches are needed. Here, we present a novel isothermal method for comprehensive characterization of protein solubility alterations using the effect on protein solubility of cations and anions in the Hofmeister series. We combine the ion-based protein precipitation approach with Proteome-Integrated Solubility Alteration (PISA) analysis and use this I-PISA assay to delineate the targets of several anticancer drugs both in cell lysates and intact cells. Finally, we demonstrate that I-PISA can detect solubility changes in minute amounts of sample, opening chemical proteomics applications to small and rare biological material.
无偏药物靶标结合去卷积和作用机制阐明是药物开发中的主要挑战。近年来，无修饰的靶标结合方法，如热蛋白质组谱分析，越来越受到关注。然而，这些方法存在局限性，因此需要新的正交方法。在这里，我们提出了一种新的等温方法，用于使用亲水分子系列中阳离子和阴离子对蛋白质溶解度变化的影响来全面表征蛋白质溶解度的改变。我们将基于离子的蛋白质沉淀方法与蛋白质组整合溶解度改变分析（PISA）相结合，并使用这种 I-PISA 测定法来描绘几种抗癌药物在细胞裂解物和完整细胞中的靶标。最后，我们证明 I-PISA 可以检测到小量样品中的溶解度变化，为化学蛋白质组学应用开辟了对小量和稀有生物材料的应用。
