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

Overview

Journal J Vis Exp

Specialties Biology
General Medicine

Date 2021 Jul 5

PMID 34223829

Citations 2

Authors

Zachary J Kirsch

Blaise G Arden

Richard W Vachet

Patanachai Limpikirati

Affiliations

Soon will be listed here.

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.

Citing Articles

Proteins Can Withstand More Extensive Labeling while Providing Accurate Structural Information in Covalent Labeling-Mass Spectrometry.

Kirsch Z, Vachet R J Am Soc Mass Spectrom. 2024; 35(5):1030-1039.

PMID: 38581471 PMC: 11167616. DOI: 10.1021/jasms.4c00043.

Site-Specific Structural Changes in Long-Term-Stressed Monoclonal Antibody Revealed with DEPC Covalent-Labeling and Quantitative Mass Spectrometry.

Gaikwad M, Richter F, Gotz R, Dorrbaum A, Schumacher L, Tonillo J Pharmaceuticals (Basel). 2023; 16(10).

PMID: 37895889 PMC: 10609731. DOI: 10.3390/ph16101418.

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