CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Anal Chem. 2020 Oct 20;92(20):13912-13921. doi: 10.1021/acs.analchem.0c02756. Epub 2020 Sep 28.
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. The MAPS method exploits the principle that drug-bound proteins will be more resistant to thermal unfolding similar to the classic TPP method, but the process of protein precipitation is assisted by microparticles. Upon heating, proteins unfold and aggregate on the surface of the microparticles. The introduction of a microparticle simplifies the whole sample preparation workflow. The proteins that precipitate on the microparticles are subjected to washing, alkylation, and digestion. The whole sample preparation is processed conveniently on the surface of the microparticles without any transfer. With the assistance of microparticles, sample loss is minimized. The MAPS method is compatible with minute amounts of initial proteins. MAPS was applied to screen the targets of several well-studied drugs and the known target proteins were successfully identified with high confidence and specificity. To investigate the specificity of the method, MAPS was applied to screen the targets of the pan-kinase inhibitor, staurosporine, and 32 protein kinases (specificity of 80%) were identified using only 20 μg of initial proteins of each sample. MAPS is an unbiased robust method for drug target screening, filling the vacancy of stability-based target screening using a precipitate.
虽然热蛋白质组谱分析（TPP）通过分析高温处理的蛋白质组样品的可溶部分在药物靶标筛选领域大放异彩，但对应的不可溶沉淀在很长一段时间内都被忽视了。沉淀的分析受到低效的样品处理程序的阻碍。在此，我们提出了一种新的方法，称为微粒辅助沉淀筛选（MAPS），用于药物靶标鉴定。MAPS 方法利用了药物结合蛋白将更能抵抗热展开的原理，与经典的 TPP 方法类似，但蛋白质沉淀的过程是由微粒辅助的。加热时，蛋白质在微粒表面展开并聚集。微粒的引入简化了整个样品制备工作流程。沉淀在微粒上的蛋白质经过洗涤、烷基化和消化处理。整个样品制备过程在微粒表面上方便地进行，无需任何转移。在微粒的辅助下，最大限度地减少了样品的损失。MAPS 方法适用于少量的初始蛋白质。MAPS 被应用于筛选几种研究充分的药物的靶标，并用高置信度和特异性成功鉴定了已知的靶标蛋白。为了研究该方法的特异性，MAPS 被应用于筛选泛激酶抑制剂 staurosporine 的靶标，仅使用每个样品 20μg 的初始蛋白质就鉴定出了 32 种蛋白激酶（特异性为 80%）。MAPS 是一种用于药物靶标筛选的无偏稳健的方法，填补了基于稳定性的沉淀靶标筛选的空白。
