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Abstract

Chemistry

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Published: June 15th, 2021

DOI:

10.3791/61983

1Department of Chemistry, University of Massachusetts Amherst, 2Molecular and Cellular Biology Program, University of Massachusetts Amherst, 3Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University

* These authors contributed equally

Abstract

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.

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Keywords Covalent Labeling

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