Abstract
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.
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