Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein–Drug Interactions,Analytical Chemistry

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Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein–Drug Interactions
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-07-11 00:00:00 , DOI: 10.1021/acs.analchem.8b01772
He Meng ₁ , Renze Ma ₁ , Michael C. Fitzgerald ₁

Affiliation

Described here is a mass spectrometry-based proteomics approach for the large-scale analysis of protein–drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔG_f) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein–drug binding affinities. This is accomplished in a chemical denaturant-induced unfolding experiment where the folded and unfolded protein fractions in each denaturant containing buffer are quantified by the amount of soluble or precipitated protein (respectively) that forms upon abrupt dilution of the chemical denaturant and subsequent centrifugation of the sample. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast. The technique was also used to identify protein targets of sinefungin, a broad-based methyltransferase inhibitor, in a human MCF-7 cell lysate. The CPP technique also yielded dissociation constant (K_d) measurements for these well-studied drugs that were in general agreement with previously reported K_d or IC₅₀ values. In comparison to a similar energetics-based technique, termed stability of proteins from rates of oxidation (SPROX), the CPP technique yielded significantly better (∼50% higher) proteomic coverage and a largely reduced false discovery rate.

中文翻译：

化学变性和蛋白质沉淀方法用于蛋白质和药物相互作用的发现和定量

这里描述的是基于质谱的蛋白质组学方法，用于蛋白质-药物相互作用的大规模分析。该方法涉及配体诱导的蛋白质折叠自由能变化（ΔΔ的评价ģ _˚F）使用化学变性和蛋白质沉淀（CPP）来确定药物的蛋白质靶标并量化蛋白质与药物的结合亲和力。这是在化学变性剂诱导的展开实验中完成的，在该实验中，每种变性剂中含有的缓冲液中折叠和未折叠的蛋白质部分均通过化学稀释剂的突然稀释和随后的离心分离后形成的可溶性或沉淀蛋白质的量来定量。样本。在此处进行的原理验证研究中，CPP技术能够鉴定出酵母菌中环孢菌素A和格尔德霉素的众所周知的蛋白质靶标。该技术还用于鉴定人MCF-7细胞裂解液中西芬芬净（一种广泛的甲基转移酶抑制剂）的蛋白质靶标。CPP技术还产生了解离常数（这些经过充分研究的药物的K _d）测量值与先前报道的K _d或IC ₅₀值总体上一致。与类似的基于能量学的技术（称为氧化稳定性蛋白质）相比，CPP技术产生的蛋白质组学覆盖率明显更高（提高了约50％），并且大大降低了错误发现率。

更新日期：2018-07-11

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