The authors acknowledge support from the National Institutes of Health (NIH) under Grant R01 GM075092. The Thermo Orbitrap Fusion mass spectrometer used to acquire some of the data described here was acquired with funds from the National Institutes of Health grant S10OD010645.

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.
<ol>
	<li>Prepare all reagent solutions in 1.5 mL microcentrifuge tubes.</li>
	<li>Prepare a protein solution of desired concentration, usually in the range of tens of &#181;M, in a 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 7.4. Alternatively, prepare a buffer-excha...

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 ...

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 of poor crystallization behavior, limited protein availability, excessive sample heterogeneity, or molecular weight limitations. Consequently, newer analysis methods have emerged that overcome these limitations. Among the emerging techniques that can provide protein structural information is mass spectrometry.
Mass spectrometry (MS) measures a molecule&#39;s mass-to-charge (m/z) ratio, so protein higher-order structural information must be obtained by encoding the desired structural information into the mass of the protein. Several approaches to encode this information have been developed, including hydrogen-deuterium exchange (HDX)1,2,3,4, chemical crosslinking (XL)5,6, and covalent labeling (CL)7,8,9,10. In HDX, backbone amide hydrogens are exchanged by slightly more massive deuteriums at rates that depend on solvent accessibility and H-bonding extent. The extent of HDX can be localized by rapidly digesting the protein into peptide fragments before separating and measuring these fragments by the mass spectrometer or by dissociating the protein in a top-down experiment. Determining the rate of exchange provides further insight into protein dynamics. HDX has proven to be a valuable tool for characterizing protein structure despite challenges associated with back exchange and the need for specialized equipment to maximize reproducibility. In XL-MS, proteins are reacted with bi-functional reagents that covalently link adjacent residue side chains within a given protein or between two proteins. In doing so, XL-MS can provide distance constraints that can be used to characterize protein structure. The regions of the protein that are cross-linked can be identified by proteolytic digestion followed by liquid chromatography (LC)-MS analysis. While XL-MS is a versatile tool that has been used to study a variety of protein complexes, including inside cells, identification of the XL products is challenging and requires specialized software.
CL-MS has emerged recently as a complementary and sometimes alternative MS-based tool to study protein structure and interactions. In CL-MS, a protein or protein complex is covalently modified with a mono-functional reagent that can react with solvent-exposed side chains (Figure 1). By comparing the modification extents of a protein or protein complex under different conditions, conformation changes, binding sites, and protein-protein interfaces can be revealed. After the CL reaction, site-specific information, often at the single amino-acid level, can be obtained using typical bottom-up proteomics workflows in which proteins are proteolytically digested, peptide fragments are separated by LC, and modified sites are identified using tandem MS (MS/MS). The rich history of bioconjugate chemistry has made numerous reagents available for CL-MS experiments. CL reagents fall into two general categories: (i) specific and (ii) non-specific. Specific reagents react with a single functional group (e.g., free amines)8,10 and are easy to implement, but they tend to provide limited structural information. Non-specific reagents react with a wide range of side chains, but often require specialized equipment such as lasers or synchrotron sources to produce these highly reactive species. Hydroxyl radicals are the most commonly used non-specific reagent, having been applied in hydroxyl radical footprinting (HRF)7,11,12,13 experiments to study a wide range of proteins and protein complexes under a variety of conditions.
Our research group has successfully used another relatively non-specific reagent called diethylpyrocarbonate (DEPC) to study protein structure and interactions in the context of CL-MS experiments14,15,16,17,18,19,20,21,22,23,24,25. DEPC offers the simplicity of specific labeling reagents (i.e., no specialized equipment is necessary to perform the reactions), while reacting with up to 30% of amino acids in the average protein. As a highly electrophilic reagent, DEPC is capable of reacting with the N-terminus and the nucleophilic side chains of cysteine, histidine, lysine, tyrosine, serine, and threonine residues. Typically, a single product of these reactions is generated, resulting in a mass increase of 72.02 Da. This single type of product contrasts with the up to 55 different products that can be produced when proteins react with hydroxyl radicals7. Such simple chemistry facilitates identification of labeled sites.
Here, we provide protocols for using DEPC-based CL-MS to study protein structure and interactions. Details associated with reagent preparation, DEPC-protein reactions, protein digestion conditions, LC-MS and MS/MS parameters, and data analysis are described. We also demonstrate the utility of DEPC labeling by providing example results from protein-metal, protein-ligand, and protein-protein interactions as well as proteins undergoing structural changes upon heating.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>3448</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino)propanesulfonic acid</td>
			<td>Millipore Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino)propanesulfonic acid sodium salt</td>
			<td>Millipore Sigma</td>
			<td>M9381</td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap RSLC C18 Column</td>
			<td>Thermo Scientific</td>
			<td>164537</td>
			<td>300 &#956;m x 15 cm, C18, 2 &#956;m, 100 A</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A998-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethylpyrocarbonate</td>
			<td>Millipore Sigma</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade water</td>
			<td>Fisher Scientific</td>
			<td>W5-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Millipore Sigma</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized chymotrypsin</td>
			<td>ProteoChem</td>
			<td>g4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized trypsin, TPCK Treated</td>
			<td>Thermo Fisher Scientific</td>
			<td>20230</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Millipore Sigma</td>
			<td>I1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine</td>
			<td>Millipore Sigma</td>
			<td>C4706</td>
			<td></td>
		</tr>
	</tbody>
</table>

covalent labeling with diethylpyrocarbonate for studying protein higher-order structure by mass spectrometry

Characterizing a protein&#39;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&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;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 &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

Identifying DEPC modification sites and modification percentages 
Mass addition due to covalent labeling can be measured at the (a) intact protein and (b) peptide levels8,9. 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...

Watch this Scientific Journal Video about Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry at JoVE.com

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

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.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Characterizing a protein structure is essential for understanding its function. Mass spectrometry 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 structure by mass spec, specific chemical reactions are performed in solution that encode a protein structural information into its mass.One particularly effective approach is to use reagents that covalently modify solving accessible amino acids 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 described the protocols associated with use of diethyl pyrocarbonate or DEPC as a covalent labeling reagent together with mass spec 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 mass spec to obtain structural information for many different proteins. Start by preparing a buffered solution of your protein of interest at a concentration in the range of tens of micromolar.We commonly use a 10 millimolar pH 7.4 MOPS buffer. Alternatively, an existing protein solution may be buffer exchange into MOPS or another buffer if the original sample contains a buffer that has nucleophilic functional groups. It's necessary that the buffer not have nucleophilic functional groups because they will interfere with the DEPC protein reaction.In a different microtube, prepare a stock solution of 100 millimolar DEPC in dry acetonitrile. Preparing the stock solution in dry acetonitrile is necessary to prevent hydrolysis of DEPC. In a new microtube, prepare solution of one molar imidazole in HPLC grade water.In a new microtube, mix the MOPS buffer and proteins solution. To the protein and buffer add an aliquot of the DEPC stock solution making sure it's properly mix it before placing the reaction mixture into a 37 degrees Celsius water bath for one minute. The volume of the DEPC stock solution that is used should be chosen based on the final DEPC protein concentration ratio that's desired.There's no one set of DEPC and protein concentrations that will work with every protein, though the optimum DEPC concentration can be estimated based on the number of solvent accessible histidine and lysine residues present in the protein. It should be noted that the volume of acetonitrile added, which is effectively the volume of the DEPC solution added should not exceed 1%of the total reaction volume as to avoid perturbation of the protein structure during the reaction. The reaction time is ultimately up to the user, although a one minute reaction under the example condition minimizes over labeling of the protein and hydrolysis of the DEPC.After one minute has passed, quench the reaction by adding the imidazole to scavenge the remaining on reacted DEPC. Making sure the final concentration of imidazole is 50 times the DEPC concentration. To identify the residues that have been modified by DEPC, the protein must be proteolytically digested into peptide fragments.For the digestion, choose conditions that are amenable to protein of interest. Common steps that are described here involve unfolding protein and then reducing and alkylating disulfide bonds so that the digestion efficiency can be improved. In our experience, unfolding the protein with a denaturant and heat prior to digestion improves the efficiency of the digestion.While protein is being unfolded, compare solutions of TCEP and iodoacetamide in an appropriate digestion buffer. TCEP should be used as the reducing agent instead of dithiothreitol or DTT because DTT can react with DEPC modified residues. Alkylation of the resulting files after reduction is essential to avoid label scrambling in which free files and the protein react with modified amino acids.Once the unfolding step has finished, reduce disulfide bonds by adding TCEP to the reaction mixture and let it react for three minutes at room temperature. After reduction, add iodoacetamide to the reaction mixture, let it react for 30 minutes at room temperature in the dark. Achieved here by placing the reaction mixture in a box.The final concentration of iodoacetamide should be double the concentration used for TCEP or 80 times the protein concentration or disulfide bond. Prepare the enzyme of interest, usually trypsin or chymotrypsin according to the manufacturer's specifications. After preparing the enzyme of choice, begin digestion and incubate the reaction mixture at 37 degrees Celsius.A 10:1 protein to enzyme ratio with a three hour digestion using immobilized enzymes is typically a good choice for DEPC labeled proteins. After digestion, the sample should either be immediately analyzed by LC-MS/MS or flash frozen with liquid nitrogen to minimize degradation of the sample and label loss. Standard LC-MS/MS parameters for bottom up proteomics can be used to identify labeled sites on the proteolytic peptide fragments.The reverse phase C18 stationary phase should be used to achieve the best separation of peptides. In our experience, capillary LC provides more reliable quantitative information about modification levels than nano LC because of the higher sample amounts used. A typical LC mobile phase that is used to separate DEPC labeled peptides is composed of two solvents.Solvent A is water with 0.1%formic acid and solvent B is a acetonitrile with 0.1%formic acid. A gradient elution is used and the separation time can be optimized based on sample complexity. A mass spectrometer capable of doing online LC-MS and MS/MS is required to identify DEPC modification sites on the peptides.In our experiments, we have successfully used several types of mass spectrometers. We have found that a Thermo Orbitrap Fusion provides excellent results, although any mass spectrometer capable of automatically performing MS/MS of many peptides during the course of an LC-MS analysis should be suitable. HPLC conditions and mass spectrometric parameters should be set properly prior to the measurement.If the sample has been flash frozen, it should be thawed right before analysis. Try your best to minimize the time between sample preparation and HPLC injection. Load and inject the digested labeled protein sample into the LC system and start the LC-MS/MS acquisition or DEPC label site identification and peak area quantification, multiple methods exist.On Thermo Fisher instruments, excalibur or proteome discoverer maybe used. Set appropriate parameters for peptide identification in a program. DEPC addition and carbamidomethylation are included as variable modifications assisting residues or allocated.Chromatographic peak areas of modified and unmodified versions of the peptides are used to determine residue level modification percentages. DEPC labeling with MS detection is also a valuable tool for characterizing higher order structural changes to proteins. From our study, DEPC covalent labeling can identify specific protein regions that undergo structural changes upon thermal and oxidative stress.In the figure, significant changes in modification percentages are shown in blue for the decrease in labeling and red for the increase in labeling, while residues with no significant labeling changes are shown in pale green. For example, after beta-2 microglobulin is exposed to heat stress, many residues that undergo significant decreases in labeling extents, N-terminus, serine 28, histidine 31, serine 33, serine 55, serine 57, and lysine 58 are closer than one side of the protein suggesting that this region of the protein undergoes a confirmational change or possibly mediates aggregation. After the protein is exposed to oxidative stress, different residues with a decrease in labeling, serine 11, histidine 13, lysine 19, lysine 41, and lysine 94 from a cluster on another phase of the protein indicating that the oxidation induced confirmational change occurs elsewhere.This study has important implications for protein therapeutics, which are currently, the fastest growing segment of the pharmaceutical market. Other work from our group has also shown that DEPC covalent labeling mass spec can detect and identify sites of confirmational changes in stressed monoclonal antibody therapeutics. As you can see, covalent labeling with DEPC is simple to implement experimentally, yet can provide valuable structural information for proteins.The structural resolution provided by DEPC labeling mass spec is modest when compared with the techniques like x-ray crystallography and NMR, but it is amenable to almost any protein regardless of its size or ability to be crystallized. DEPC covalent labeling mass spec also works with protein mixtures and can work with very complicated sample types, such as cell lysate and it haCat cells. After watching this video, we are confident that you will find DEPC labeling a useful tool for characterizing protein structure.

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

Abstract