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.
2. Covalent labeling of intact protein
3. Preparation of protein digest for bottom-up LC-MS
NOTE: Choose digestion conditions that are amenable to the protein of interest. Common steps involve unfolding the protein and reducing and alkylating any disulfide bonds.
4. LC-MS/MS Analysis
NOTE: Standard LC-MS/MS parameters for bottom-up proteomics can be used to identify labeled sites on the proteolytic peptide fragments. A general example is outlined below.
5. Data analysis
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 performed at the peptide level. After the labeling and quenching steps, the labeled proteins are subjected to bottom-up proteomic analysis (i.e., disulfide reduction, alkylation, proteolytic digestion, and LC-MS/MS). Figure 3 shows how the DEPC labeled sites are identified and their modification levels are calculated for a DEPC CL-MS experiment on the protein β-2-microglobulin (β2m)21. MS/MS is used to sequence the labeled peptides and pinpoint their DEPC labeled sites, while modification percentages are calculated from their relative peak areas in extracted ion chromatograms (See Figure 3A).
Protein surface mapping using DEPC CL-MS
Because of the relationship between protein topology and labeling rate, DEPC has been used to study changes in protein higher-order structure (HOS) and identify protein interaction sites8,9. An example is the effect of Cu(II) binding on the structure of β2m. DEPC modification rate coefficients of peptide fragments from unbound β2m and Cu(II)-bound β2m (b2m-Cu) can be measured (Table 2) by varying DEPC concentrations and generating second-order kinetic plots14,24. The DEPC reactivity of residues in β2m-Cu reveals significant labeling rate changes for His31, Ser33, and Thr4, while other sites, including different His residues, are statistically unchanged. These data are consistent with the fact that His31, but not other His residues in β2m, binds Cu causing structural changes near His31 (i.e., Ser33) and near the N-terminus (i.e., Thr4) (Figure 4)14,26,27.
DEPC CL-MS for identifying changes in HOS
DEPC labeling with MS detection is also a valuable tool for characterizing HOS changes to proteins, which has important implications for protein therapeutics, which are currently the fastest growing segment of the pharmaceutical market28. DEPC CL can identify specific protein regions that undergo structural changes upon thermal and oxidative stress18. After β2m is exposed to heat stress, many residues that undergo significant decreases in labeling extents (N-terminus, Ser28, His31, Ser33, Ser55, Ser57, Lys58) are clustered on one side of the protein, suggesting that this region of the protein undergoes a conformational change or possibly mediates aggregation (Figure 5A). In addition to these residues, after the protein is exposed to oxidative stress, other residues with a decrease in labeling (Ser11, His13, Lys19, Lys41, Lys94) form a cluster on another face of the protein, indicating that the oxidation-induced conformational changes occur elsewhere (Figure 5B). Other work from our group has also shown that DEPC CL-MS can detect and identify sites of conformational changes in heat-stressed monoclonal antibody therapeutics20.
DEPC CL-MS to study protein-protein interactions
Insight into protein-protein interaction sites and aggregation interfaces for amyloid-forming proteins can be obtained using DEPC labeling as well9. Decreases in the modification levels of residues upon oligomer formation can reveal the binding interfaces. DEPC CL-MS was used to characterize the pre-amyloid oligomers of β2m, which is the protein that forms amyloids in dialysis-related amyloidosis29, after initiating amyloid formation with Cu(II)15,16,26. Comparing the DEPC reactivity of the β2m monomer with the pre-amyloid β2m dimer that is formed 2 h after adding Cu(II) shows that nine residues undergo decreases in labeling while six residues undergo no change or slight increases in labeling (Figure 6A). Upon mapping these changes on the β2m, it is immediately apparent that the dimer interface involves the ABED β-sheets of β2m monomers (Figure 6B)15.
DEPC CL-MS to study protein-ligand binding
Ligand binding to proteins leads to decreases in the solvent accessibility of residues that interact with the ligand, and DEPC-based CL-MS can be used to identify ligand-binding sites on proteins. For example, the binding sites of epigallocatechin-3-gallate (EGCG) on β2m under amyloid-forming conditions can be identified by significant decreases in DEPC labeling at the residues buried by EGCG binding. In the presence of ECGC, Lys6 and Lys91 have lower DEPC modification percentages (Figure 7), and these residues are clustered in one region of the protein, indicating residue protection due to ligand binding. The N-terminus, Thr4, and His31, meanwhile, undergo increases in labeling extents (Figure 7), which are indicative of ECGC-induced structural changes and suggest that the Cu(II) binding sites on β2m (N-terminus and His31)14,30,31 may be disrupted as a result of ECGC binding32. In addition, the binding sites of the other two small-molecule inhibitors of β2m amyloid formation, rifamycin SV and doxycycline, have been identified using CL-MS19. In this study, however, results from DEPC labeling alone were not enough to map the binding sites with sufficient structural resolution. Results from three different CL reagents, namely DEPC, BD, and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide – glycine ethyl ester (EDC/GEE) pair were necessary to better pinpoint the binding sites19.
Improving structural resolution and reducing label scrambling
Even though DEPC labeling causes the formation of a covalent bond, label loss due to hydrolysis can occur, especially for Ser, Thr, and Tyr residues (Figure 8). Label loss can be minimized by decreasing the time between DEPC CL reaction and LC-MS analysis using short proteolytic digestions (e.g., 2 h digestion with immobilized enzymes) rather than overnight digestions. Fast digestions result in more modified residues being measured, increasing the amount of protein structural information. For example, a 2 h digestion with immobilized chymotrypsin consistently leads to the identification of more DEPC-modified residues in β2m (Figure 9)17. With an overnight digestion about 75% of the Ser, Thr, Tyr, His, and Lys residues in β2m are detected, but when the time between labeling and LC-MS is reduced by using a 2 h digestion, 95% of these residues in β2m are detected. Most of the newly detected modified residues are Tyr and Thr residues that are prone to hydrolysis.
Over the course of our work with DEPC, we have noticed that in some proteins, Cys residues that from disulfide bonds are modified by DEPC, even though the disulfide bonds are intact during the DEPC labeling reactions. Such labeling of Cys residues occurs because free thiols can react with other modified residues in solution (Figure 10), leading to so-called "label scrambling" as the carbethoxy group is transferred to the Cys residue. Label scrambling decreases the modification levels at other residues and provides incorrect protein structural information. To avoid label scrambling, the free Cys thiols must be fully alkylated right after disulfide reduction33.
Figure 1: Covalent labeling-mass spectrometry (CL-MS). A monofunctional reagent is used to modify solvent accessible amino acids and can be used to provide site-specific information about conformational changes or interaction interfaces in proteins (top vs. bottom image). The modified protein is proteolytically digested, and the resulting peptides are analyzed by liquid chromatography (LC) in conjunction with MS and tandem MS (MS/MS). Figure has been adapted from Limpikirati, P., Liu, T., Vachet, R. W. Covalent Labeling-Mass Spectrometry for Studying Protein Structure and Interactions. Methods 144, 79-93 (2018). Please click here to view a larger version of this figure.
Volume Used (μL) | Concentration in Solution (μM) | |
Protein (100 μM, MOPS) | 50 | 50 |
MOPS (10 mM, pH 7.4) | 48.8 | n/a |
DEPC (100 mM) | 0.2 | 200 (=4x[protein]) |
Imidazole (1 M) | 1 | 10,000 (=50x[DEPC]) |
Total Volume | 100 |
Table 1. General DEPC labeling protocol.
Figure 2. Example LC gradient for separation of peptides. Please click here to view a larger version of this figure.
Figure 3 Illustration of how DEPC labeled sites are identified and their modification levels are calculated. After DEPC labeling and proteolytic digestion, (A) LC-MS analysis of the digested protein is performed. Peak areas of (B) unlabeled and (C) labeled peptides in a chromatogram are used to calculate the labeling percentage. During LC-MS, peptides are subjected to CID MS/MS. Tandem mass spectra of (D) unlabeled and (E) & (F) labeled peptides obtained at specific retention times are used for peptide sequencing and identification of DEPC labeled sites. Please click here to view a larger version of this figure.
Residue | b2m | b2m-Cu |
Thr4 | 0.082 ± 0.004 | 0.052 ± 0.009 |
His13 | 0.041 ± 0.003 | 0.033 ± 0.005 |
His31 | 0.010 ± 0.001 | 0.003 ± 0.001 |
Ser33 | 0.010 ± 0.002 | 0.004 ± 0.001 |
His51 | 0.036 ± 0.003 | 0.030 ± 0.004 |
Ser88 | 0.029 ± 0.007 | 0.020 ± 0.006 |
Table 2 Site-specific DEPC modification rate coefficients (k, M-1s-1) for b2m in the absence and presence of Cu(II).
Figure 4 Using DEPC CL-MS to determine the effect of Cu(II) binding on the structure of β2m: (A) Protein surface mapping of the residues with significant decreases in DEPC labeling rates (blue), indicating changes in their solvent accessibility upon Cu(II) binding, and those with no significant changes in labeling rate (magenta) (PDB accession code 1JNJ). (B) Example site-specific second-order kinetic plots of the reaction of β2m with different concentrations of DEPC in the presence and absence of Cu(II). The labeling rate coefficient (k) can be obtained from the slope of the kinetic plot. Please click here to view a larger version of this figure.
Figure 5 Covalent labeling results for stressed vs. native β2m: (A) Heat stress – heating at 75 °C for 24 h and (B) oxidative stress – with 3% H2O2 for 24 h. Significant changes in modification percentages are shown in blue (decrease in labeling) and red (increase in labeling) while residues with no significant labeling changes are shown in pale green (PDB accession code 1JNJ). Please click here to view a larger version of this figure.
Figure 6 Using DEPC CL-MS to determine the dimer interface for the pre-amyloid dimer of β2m: (A) Summary of DEPC modification level changes for modified residues in the monomer (i.e., t = 0) and dimer 2 h after adding Cu(II). Residues with significant decreases in labeling extent are denoted by an asterisk (*). (B) Covalent labeling results mapped on the β2m structure. Residues with decreased labeling are shown in blue and residues with no changes or slight increases in labeling are shown in red (PDB accession code 1LDS). Please click here to view a larger version of this figure.
Figure 7 Covalent labeling results for EGCG-bound vs. unbound b2m. The DEPC modification percentages are shown for the displayed residues. Statistically significant differences in the covalent labeling percentage are denoted by an asterisk (*). Increases in labeling upon ECGC binding are shown in red while decreases are shown in blue. Please click here to view a larger version of this figure.
Figure 8 DEPC covalent labeling reactions (nucleophilic acyl substitution) and label loss (hydrolysis of carbethoxylated residues) Please click here to view a larger version of this figure.
Figure 9 Mapping of the measured modification sites on β2m. (A) Labeled sites after conventional overnight digestion, and (B) labeled sites after a 2 h digestion with immobilized chymotrypsin. Please click here to view a larger version of this figure.
Figure 10 Hypothesized mechanism of DEPC label scrambling (Cys capture of carbethoxylated His) Please click here to view a larger version of this figure.
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 |
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.
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.
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.