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

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.