CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2023 Sep 19;95(37):13779-13787. doi: 10.1021/acs.analchem.3c00072. Epub 2023 Sep 7.
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. Here, we develop an iso-pH shift assay (ipHSA) method, a proteome-wide target identification method that detects ligand-induced protein solubility shifts by precipitating proteins with a single concentration of acidic agent followed by protein quantification via data-independent acquisition (DIA). Using a pan-kinase inhibitor, staurosporine, we demonstrated that ipHSA increased throughput compared to the previously developed pH-dependent protein precipitation (pHDPP) method. ipHSA was found to have high complementarity in staurosporine target identification compared with the improved isothermal shift assay (iTSA) and isosolvent shift assay (iSSA) using DIA instead of tandem mass tags (TMTs) for quantification. To further improve target identification sensitivity, we developed an integrated protein solubility shift assay (IPSSA) by pooling the supernatants yielded from ipHSA, iTSA, and iSSA methods. IPSSA exhibited increased sensitivity in screening staurosporine targets by 38, 29, and 38% compared to individual methods. Increasing the number of replicate experiments further enhanced the sensitivity of target identification. Meanwhile, IPSSA also improved the throughput and reduced the cost compared with previous methods. As a fast and efficient tool for drug target identification, IPSSA is expected to have broad applications in the study of the mechanism of action.
目标蛋白通常在与配体结合后变得更加稳定，从而更能抵抗变性。基于这一现象，已经开发出了几种无需共价修饰配体的方法来鉴定特定配体的靶蛋白。这些方法通常采用复杂的工作流程，成本高，通量有限。在这里，我们开发了一种等 pH 漂移分析（ipHSA）方法，这是一种基于全蛋白质组的靶蛋白鉴定方法，通过用单一浓度的酸性试剂沉淀蛋白质来检测配体诱导的蛋白质溶解度漂移，然后通过数据非依赖性采集（DIA）进行蛋白质定量。使用泛激酶抑制剂星形孢菌素，我们证明了 ipHSA 比以前开发的 pH 依赖性蛋白质沉淀（pHDPP）方法具有更高的通量。与使用 DIA 代替串联质量标签（TMT）进行定量的改进等温漂移分析（iTSA）和等溶剂漂移分析（iSSA）相比，ipHSA 在星形孢菌素靶蛋白鉴定方面具有更高的互补性。为了进一步提高靶蛋白鉴定的灵敏度，我们通过汇集 ipHSA、iTSA 和 iSSA 方法的上清液，开发了一种集成蛋白质溶解度漂移分析（IPSSA）。与单独的方法相比，IPSSA 在筛选星形孢菌素靶蛋白时的灵敏度分别提高了 38%、29%和 38%。增加重复实验的次数进一步提高了靶蛋白鉴定的灵敏度。同时，与以前的方法相比，IPSSA 还提高了通量并降低了成本。作为一种快速有效的药物靶蛋白鉴定工具，IPSSA 有望在作用机制研究中得到广泛应用。
