Department of Chemistry, University of Massachusetts Amherst;
Department of Chemistry, University of Massachusetts Amherst; Molecular and Cellular Biology Program, University of Massachusetts Amherst;
J Vis Exp. 2021 Jun 15(172). doi: 10.3791/61983.
Characterizing a protein's higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein's structure by MS, specific chemical reactions are performed in solution that encode a protein's structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as β2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.
对蛋白质的高级结构进行特征分析对于理解其功能至关重要。质谱（MS）已成为一种非常有用的工具，尤其适用于传统方法难以研究的蛋白质系统。为了通过 MS 研究蛋白质的结构，可以在溶液中进行特定的化学反应，将蛋白质的结构信息编码到其质量中。一种特别有效的方法是使用共价修饰可及氨基酸侧链的试剂。这些反应导致质量增加，当与蛋白水解酶消化和串联质谱结合使用时，可以以残基分辨率进行定位。在这里，我们描述了使用焦碳酸二乙酯（DEPC）作为共价标记试剂并结合 MS 检测的相关方案。DEPC 是一种高亲电分子，能够标记平均蛋白质中多达 30%的残基，从而提供出色的结构分辨率。DEPC 已成功与 MS 一起用于获得β2-微球蛋白等小单域蛋白质以及单克隆抗体等大的多域蛋白质的结构信息。
