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

Summary

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

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.

Introduction

Proteins are essential biomolecules in virtually every physiological process. The variety of functions that proteins perform are possible because of the structures they adopt and the interactions that they have with other biomolecules. To understand protein function at a deeper level, biochemical and biophysical tools are needed to elucidate these important structural features and interactions. Traditionally, X-ray crystallography, cryogenic electron microscopy, and nuclear magnetic resonance (NMR) spectroscopy have provided the desired atomic-level detail to reveal protein structure. However, numerous protein systems cannot be interrogated by these techniques because....

Protocol

1. Protein and reagent preparation

NOTE: This protocol includes an example workflow for labeling a protein with DEPC. Some conditions and reagent concentrations listed may vary based on the protein of choice.

1. Prepare all reagent solutions in 1.5 mL microcentrifuge tubes.
2. Prepare a protein solution of desired concentration, usually in the range of tens of µM, in a 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 7.4. Alternatively, prepare a buffer-exchange existing protein solution in 10 mM pH 7.4 MOPS if the sample contains a nucleophilic buffer that would be reactive with DEPC. Other buffers (e.g.,....

Results

Identifying DEPC modification sites and modification percentages
Mass addition due to covalent labeling can be measured at the (a) intact protein and (b) peptide levels^8,9. At the intact level, a distribution of protein species with different numbers of labels can be obtained from direct analysis or LC-MS of labeled protein samples. To obtain higher resolution structural information (i.e., site-specific labeling data), measurements must be performe.......

Discussion

Critical Steps
Several points regarding experimental design should be considered to ensure reliable labeling results. First, to maximize protein labeling, it is necessary to avoid buffers with strongly nucleophilic groups (e.g., Tris) because they can react with DEPC and lower the extent of labeling. It is also conceivable that such buffers could react with labeled residues, causing the removal of the label and therefore loss of structural information. We recommend MOPS as a buffer, but phosphate buffered .......

Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tube	Thermo Fisher Scientific	3448
3-(N-morpholino)propanesulfonic acid	Millipore Sigma	M1254
3-(N-morpholino)propanesulfonic acid sodium salt	Millipore Sigma	M9381
Acclaim PepMap RSLC C18 Column	Thermo Scientific	164537	300 μm x 15 cm, C18, 2 μm, 100 A
Acetonitrile	Fisher Scientific	A998-1
Diethylpyrocarbonate	Millipore Sigma	D5758
HPLC-grade water	Fisher Scientific	W5-1
Imidazole	Millipore Sigma	I5513
Immobilized chymotrypsin	ProteoChem	g4105
Immobilized trypsin, TPCK Treated	Thermo Fisher Scientific	20230
Iodoacetamide	Millipore Sigma	I1149
Tris(2-carboxyethyl)phosphine	Millipore Sigma	C4706