Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas.
Chem Biol Drug Des. 2019 Jun;93(6):1158-1165. doi: 10.1111/cbdd.13403. Epub 2018 Oct 22.
Defining molecular targets of alcohol and understanding the molecular mechanism of alcohol actions are necessary to develop effective therapeutics for alcohol use disorder (AUD). Here, we describe a detailed protocol for identifying alcohol-binding site(s) in proteins using diazirine-based azialcohol as photoaffinity labeling agents. Upon photoirradiation, azialcohol photoincorporates into alcohol-binding proteins. The stoichiometry and site of azialcohol photoincorporation can be determined using high-resolution mass spectrometry. Identification of the alcohol-binding residues in protein followed by measuring the biological significance of these residues in regulating alcohol action are important steps in characterizing the molecular targets of alcohol.
定义酒精的分子靶点，并了解酒精作用的分子机制，对于开发治疗酒精使用障碍（AUD）的有效疗法是必要的。在这里，我们描述了一种使用重氮醇作为光亲和标记试剂来鉴定蛋白质中酒精结合位点的详细方案。光照射后，重氮醇会掺入到酒精结合蛋白中。通过高分辨率质谱可以确定重氮醇光掺入的化学计量和位置。在确定蛋白质中的酒精结合残基后，测量这些残基在调节酒精作用中的生物学意义是表征酒精分子靶点的重要步骤。
