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Differential Protein Labeling Based on Electrochemically Generated Reactive Intermediates Lars Büter,†,‡ Helene Faber,‡ Tina Wigger,†,‡ Martin Vogel,‡ and Uwe Karst*,†,‡ †

Westfälische Wilhelms-Universität Münster, NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany ‡ Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Corrensstraße 30, 48149 Münster, Germany

Downloaded by UNIV OF SOUTH DAKOTA on September 10, 2015 | http://pubs.acs.org Publication Date (Web): September 9, 2015 | doi: 10.1021/acs.analchem.5b02497

S Supporting Information *

ABSTRACT: A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as β-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments. n the field of proteomics, the identification as well as the quantification of proteins in complex mixtures is required in order to determine the state of an organism.1 The specification of the proteome at a given point of time may yield information about the state of an organism and may allow the recognition of possible diseases in time. The unambiguous identification of proteins is preferably performed via the identification of a specific peptide after enzymatic digestion, which can be carried out, e.g., with the protease trypsin. The determination of the number of a certain amino acid in a protein provides additional information and therefore allows a simplified identification of the corresponding protein by means of mass fingerprinting.2 Moreover, the cysteine content reveals additional information such as on the structure, site accessibility, and activity of the investigated proteins. The amino acid cysteine is available in around 90% of all proteins and therefore particularly suitable for these investigations.3 Cysteine is the only amino acid containing a nucleophilic thiol group, which is an appropriate target for selective tagging with electrophilic compounds such as electrochemically generated reactive intermediates. Electrochemistry (EC) in combination with liquid chromatography (LC) and mass spectrometry (MS) is increasingly applied in protein investigations. In drug metabolism studies, electrochemical methods can be used to in vitro simulate the oxidative drug metabolism.4−9 Upon electrochemical oxidation,

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it is possible to generate reactive drug intermediates in order to perform protein binding studies under defined conditions.10 Tryptic digestion of the generated protein adducts and identification of the modified tryptic peptides enables the determination of the binding site.10−15 Moreover, electrochemical oxidation can be applied for proteolysis.16−20 Since EC can be performed online and no further purification of the electrochemically digested sample is required prior to LC/MS analysis, this alternative approach offers several advantages compared to the commonly carried out enzymatic digestion procedures. In further studies, it was shown that the application of negative potentials allows the electrochemical reduction of disulfide bonds in peptides and proteins.21−27 As EC is capable of generating reactive electrophiles, which show specific reactivity toward thiol groups, EC/MS is a promising tool to perform specific tagging of cysteine residues in peptides and proteins. Girault and co-workers developed a method for the generation of benzoquinone tags, which were formed upon dehydrogenation of hydroquinone.28−36 In their studies, they electrochemically oxidized hydroquinone on a microfabricated polymer microspray chip with an integrated Received: July 3, 2015 Accepted: September 1, 2015

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DOI: 10.1021/acs.analchem.5b02497 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

obtain reactive NAPQI (N-acetyl-p-benzoquinone imine) intermediates. All studies performed were focused on the structural elucidation of proteins by identifying the number and localization of cysteine residues.

carbon microelectrode to which the ESI voltage was applied. Thus, they took advantage of the inherent electrochemistry during electrospray ionization, which previously had been investigated extensively by Van Berkel et al.37−41 Girault and co-workers compared their homemade nanospray interface with a classic ESI probe,28 elucidated the tagging mechanism,29 characterized the influence of the microspray design on the tagging process,30 and determined the optimum probe for electrochemical tagging of cysteine residues.31,34 Finally, the developed method was successfully applied in protein identification studies.34 Van Berkel and Kertesz also made use of the ESI inherent electrochemistry and replaced the commonly used stainless steel electrodes of an ESI interface by porous flow-through electrodes. Therefore, they were able to simultaneously reduce a disulfide containing peptide at the upstream grounding point, while benzoquinone tags were generated at the emitter electrode.42 Furthermore, Van Berkel et al. utilized a liquid microjunction surface sampling probe (LMJ-SSP) for cysteine tagging. A peptide containing two cysteine residues was sampled on the surface of a conductive glassy carbon electrode, labeled with electrochemically generated benzoquinone intermediates, extracted from the surface, and directly analyzed by means of MS.43 A recent review summarizes applications in the field of protein adduct formation caused by electrochemically generated reactive intermediates.10 Further labeling strategies of cysteine residues for MS based proteomic investigations involve different cysteine specific functional groups. Common compounds in this connection are, e.g., iodoacetyl derivatives, maleimides, acryloyl compounds, or thiol-disulfide exchange systems. Next to the information on the cysteine content in one protein, tagging of proteins can be used to improve the electrospray ionization efficiency of peptides and proteins, to perform cross-linking experiments, and to obtain quantitative information. 44 Quantification is commonly performed by using tagging agents in their native and isotopically labeled form. Thus, on the basis of the ratio of the two obtained MS signals, quantitative information can be obtained.45 Gygi et al. were the first who used the ICAT (isotope coded affinity tag) approach, which allows relative protein quantification of two different states of an organism. ICAT reagents contain a cysteine specific reactive group, an isotopically enriched linker, and an affinity tag in order to enrich cysteine containing peptides out of complex mixtures.46 Besides protein labeling for ESI-MS experiments, analogous strategies for MALDI-MS investigations were developed. Qiao et al. made use of the photooxidation processes of the UV-laser during MALDI in order to generate reactive benzoquinones, which can directly bind to biomolecules present in the sample.47,48 Ma et al. developed a dual tagging approach based on benzoquinone and methyl-pbenzoquinone in order to obtain quantitative information on cysteine containing biomolecules.49 In this study, cysteine specific labeling compounds were electrochemically generated in their native and isotopically enriched form. With this, a specific mass difference for differentially labeled biomolecules was achieved, thus enabling the easy determination of cysteine residues. For this purpose, two different systems were investigated and compared. On the one hand, phenol and 13C6-phenol were electrochemically oxidized in order to generate the respective benzoquinone intermediates. On the other hand, a system consisting of acetaminophen and D4-acetaminophen was used in order to

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EXPERIMENTAL SECTION Chemicals. Acetaminophen (APAP), glutathione (GSH), βlactoglobulin A (LGA), human serum albumin (HSA), bovine serum albumin (BSA), hemoglobin (Hb), carbonic anhydrase I (CAI), guanidine hydrochloride, 13C6-phenol, and ammonium formate were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Formic acid was ordered from Fluka Chemie (Buchs, Switzerland). Phenol was purchased from ABCR (Karlsruhe, Germany) and D4-acetaminophen from CDN Isotopes (Augsburg, Germany). Furthermore, ammonia was acquired from Merck (Darmstadt, Germany) and trypsin from bovine pancreas from Promega (Mannheim, Germany). All chemicals and solvents were used in the best quality available. Water was purified before utilization with an Aquatron A4000D system (Barloworld Scientific, Nemours, France). Electrochemical Oxidation and Detection of Acetaminophen by Means of Online EC/MS. The setup used is shown in Figure 1a. Electrochemical oxidation of APAP was

Figure 1. Instrumental online setups for the electrochemistry/(liquid chromatography/)mass spectrometry experiments carried out within this work. (a) EC/APCI-MS, (b) EC/GSH/ESI-MS, and (c) EC/ protein/LC/ESI-MS. Electrochemical oxidation was performed with a boron-doped diamond working electrode by applying a potential ramp (0−2.5 V vs Pd/H2) or a constant potential. All potentials applied were controlled by a homemade potentiostat.

performed with an amperometric thin-layer cell (Reactor Cell, Antec Leyden, Zoeterwoude, The Netherlands) equipped with a boron-doped diamond (BDD) working electrode, a graphitedoped Teflon counter electrode, and a Pd/H2 reference electrode. The potential was controlled by a homemade potentiostat. In order to detect APAP and the oxidation products, the cell was directly coupled to a high-resolution mass spectrometer (Exactive Orbitrap, Thermo Fisher Scientific, Bremen, Germany) with an atmospheric-pressure ionization (APCI) source. With a flow rate of 10 μL/min, a 50 μM solution of APAP in 10 mM aqueous ammonium formate (pH 7; adjusted with 1% ammonia solution) and acetonitrile (50:50, v/v) was pumped through the electrochemical cell, to which a potential ramp between 0 and 2.5 V versus Pd/H2 with a scan rate of 10 mV/s was applied. Mass spectra were recorded in the negative ionization mode (for detailed MS parameters, see Section SI-1). Formation and Investigation of Glutathione Adducts by Online EC/MS. In Figure 1b, the used setup is schematically shown. In order to study the adduct formation of generated reactive oxidation products with glutathione, solutions of a mixture of (a) 25 μM phenol and 13C6-phenol B


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Figure 2. Schematic overview on (a) oxidation pathway of phenol, which results in the formation of reactive benzoquinone intermediates, (b) oxidation pathway of acetaminophen, which results in the formation of the reactive species NAPQI or benzoquinone, respectively, and (c) Michaeltype addition of benzoquinone and NAPQI with glutathione resulting in the corresponding adducts.

and (b) 25 μM acetaminophen and D4-acetaminophen, respectively, were electrochemically oxidized with the same conditions as described above. However, instead of a homemade potentiostat, the potential was controlled using a Roxy potentiostat (Antec Leyden). To the effluent of the EC cell, a 50 μM solution of glutathione in 10 mM aqueous ammonium formate (pH 7; adjusted with 1% ammonia solution) was added via a T-piece with a flow rate of 10 μL/ min. The reaction mixture with a total flow rate of 20 μL/min was pumped through a reaction coil for 2 min and analyzed via electrospray ionization time-of-flight MS (micrOTOF, Bruker Daltonics, Bremen, Germany) in the negative ion mode (for detailed MS parameters, see Section SI-1). Generation and Analysis of Protein Adducts by Online EC/LC/MS. Figure 1c shows the setup applied in these experiments. A 100 μM solution of either phenol, 13C6phenol, acetaminophen, or D4-acetaminophen was electrochemically oxidized with the previously described EC setup. In order to obtain the maximum amount of reactive intermediates, the oxidations were performed at an optimized constant potential of 2.3 V (phenol, 13 C 6 -phenol) and 1.8 V (acetaminophen, D4-acetaminophen) vs Pd/H2. Via a Tpiece, a 20 μM solution of the corresponding protein (βLGA, HSA, BSA, Hb, CAI), dissolved in 6 M guanidine hydrochloride (for denaturation), was added with a flow rate of 10 μL/min. After mixing, the solution was pumped through a reaction coil for 5 min and collected in a 5 μL injection loop in a ten-port switching valve. By switching the valve to injection position, the protein solution was analyzed online by means of LC/MS. For this purpose, an Alexys LC system (Antec Leyden) and a micrOTOF-MS (Bruker Daltonics) equipped with an ESI source were used. The LC system included two LC 100 pumps, an OR 110 organizer rack with a degasser and a pulse dampener, an AS 100 autosampler, and a Roxy Potentiostat and column oven. The mixture was separated on a C5 Wide Pore Column (Discovery BIO Wide Pore C5, 150 × 2.1 mm, 5 μm particle size, 300 Å, Supelco, Steinheim, Germany) using a binary gradient of acetonitrile and 0.1% formic acid (for detailed LC parameters, see Section SI-1). In

order to avoid guanidinium hydrochloride from entering the MS, the LC effluent was discarded for the first 2 min of the separation. MS detection was performed in the positive ionization mode (for detailed MS parameters, see Section SI-1). Tryptic Digest of Protein Adducts. A solution containing a mixture of phenol and 13C6-phenol with a total concentration of 100 μM phenol was electrochemically oxidized with the previously described EC setup. To the effluent of the EC cell, a 20 μM solution of CAI was added via a T-piece, and the reaction mixture was collected in a vial for 100 min. Afterward, the solution was purified by means of size exclusion using PD midiTrap G-25 columns (GE Healthcare, Berlin, Germany) according to the supplier’s protocol. Then, ammonium bicarbonate and calcium chloride were added to the sample with a final concentration of 50 mM and 1 mM, respectively. Trypsin, which was dissolved in the supplied resuspension buffer (Promega, Mannheim, Germany), was given to the protein sample in a 1:25 (trypsin/protein) ratio, and the solution was incubated for 15.5 h at 37 °C. The tryptic digest was stopped by acidifying the sample with acetic acid to a final value of pH 3−4. Finally, 5 μL of the sample was analyzed by means of LC/ESI-HR-MS using a Shimadzu HPLC system (Duisburg, Germany) and an Exactive Orbitrap MS (Thermo Fisher Scientific). The LC system consisted of two LC10ADVP pumps, a SIL-10A autosampler, a SCL-10AVP system controller, a DGC-14A degasser, and a CTO-10ASVP column oven. The LCSolution software (version 1.2.2, Shimadzu) was used to control the system. The mixture was separated on a C5 Wide Pore Column (Discovery BIO Wide Pore C5, 150 × 2.1 mm, 5 μm particle size, 300 Å, Supelco, Steinheim, Germany) using a binary gradient of 0.1% formic acid and acetonitrile (for detailed LC parameters, see Section SI-1). In order to prevent salts from entering the MS, the LC eluent of the first 1.5 min was discarded. MS detection was performed in the positive ionization mode (for detailed MS parameters, see Section SI-1). Tryptic digest of Hb was performed accordingly (see Section SI-2). C


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RESULTS AND DISCUSSION

Electrochemical Oxidation and GSH Adduct Formation. Phenolic compounds are known to form reactive electrophiles upon electrochemical oxidation. Therefore, within this study, phenol and acetaminophen were selected as starting compounds for the electrochemical generation of cysteine specific labeling compounds. Phenol and acetaminophen were used in their native as well as in their respective isotopically labeled form (13C6-phenol and D4-acetaminophen) in order to obtain a specific mass difference for differentially labeled biomolecules. The electrochemical oxidation pathways of phenol and acetaminophen are shown in Figure 2a,b.5,50 In the first step, the oxidation of phenol results in the formation of hydroquinone. Since hydroquinone has a lower oxidation potential compared to phenol, dehydrogenation leading to the reactive benzoquinone (Q) intermediate can take place thereafter. Benzoquinone is of electrophilic nature and, therefore, a suitable compound for the selective reaction with the nucleophilic thiol functions as those of cysteine residues in biomolecules. Electrochemical oxidation of acetaminophen (APAP) leads to the generation of the dehydrogenated NAPQI intermediate, which is also a known electrophile showing a selective reactivity toward thiols. However, in aqueous media, NAPQI can alternatively be hydrolyzed. Further deamination results in the generation of benzoquinone. On the basis of their electrophilic nature, electrochemically generated benzoquinone and NAPQI are able to undergo a 1,4Michael addition with cysteine containing biomolecules like the tripeptide glutathione (GSH) as shown in Figure 2c. In the first experiments, APAP was electrochemically oxidized and the effluent of the EC cell was continuously analyzed by means of APCI-MS. The generated NAPQI intermediate contains no functional group, which can be protonated or deprotonated during ESI. Therefore, APCI is the ionization technique of choice, since it allows the ionization of quinoid structures in the negative ionization mode through electron capture processes. In order to determine the optimum oxidation potential for the generation of NAPQI, a potential ramp between 0 and 2.5 V (vs Pd/H2) was applied. The obtained mass spectra were plotted against the applied oxidation potential, thus allowing an easy identification of the optimum oxidation potential. In Figure 3a, the corresponding mass voltammogram is shown. The signal intensity of APAP (m/z 150.1) decreases, whereas the intensity of NAPQI (m/z 149.0) increases with a maximum at around 2.3 V. As a result, it could be shown that reactive intermediates can be generated with high yields at higher oxidation potentials. In order to demonstrate the reactivity of the generated reactive species toward cysteines and to determine the optimum potential for the generation of reactive intermediates, mixtures of phenol/13C6-phenol and APAP/D4-APAP were electrochemically oxidized. Again, a potential ramp from 0 to 2.5 V was applied and GSH was directly added to the effluent of the cell. After passing a reaction coil for 2 min, the reaction mixture was detected by ESI-MS in order to record mass voltammograms (Figure 3b,c). In Figure 3b, the mass voltammogram after electrochemical oxidation of phenol/13C6-phenol and adduct formation with GSH is shown. Two signals (m/z 414 and m/z 420) with increasing signal intensities starting at a potential of 1.7 V and a maximum intensity at 2.5 V were detected. On the basis of the determined

Figure 3. Mass voltammograms of (a) electrochemical oxidation of acetaminophen (m/z 150.1) leading to the generation of reactive NAPQI (m/z 149.0), (b) adduct formation of glutathione with electrochemically generated benzoquinone/13C6-benzoquinone (m/z 414.1 and 420.1), and (c) NAPQI/D4-NAPQI (m/z 455.1 and 458.1). In all cases, the generation of reactive intermediates is favored at higher oxidation potentials resulting in a maximum signal intensity of the intermediates or corresponding adducts at 2.3−2.5 V vs Pd/H2.

exact masses (see Section SI-3 for detected and calculated m/z as well as their relative deviation), the signals were identified to have emerged by the formation of GSH adducts with electrochemically generated benzoquinone and 13C6-benzoquinone. The mass difference of Δm/z 6, which can be traced back to six heavy-isotope labeled carbon atoms in 13C6-phenol, allows the resolved detection of differentially labeled glutathione. The corresponding mass voltammogram after oxidation of APAP/D4-APAP is shown in Figure 3c. In this case, two signals (m/z 455 and m/z 458) with increasing signal intensities were detected again. The signals appearing from about 1.2 V to their maximum at 2.3 V can be traced back to adducts of GSH with reactive NAPQI and D4-NAPQI. The mass difference of only three mass units (Δm/z 3) can be explained by the exchange of one deuterium atom through D


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Analytical Chemistry GSH in D4-NAPQI resulting in the 3-fold deuterium labeled GSH adduct. However, the obtained mass difference is not sufficient to detect totally resolved signals for both adducts. The combined isotope satellite of the 13C- and 34S-isotopes of the NAPQI-GSH adduct overlaps with the main signal of the deuterated adduct. Furthermore, the intensity of the 13Csatellite of the deuterated adduct is slightly higher than expected. This can be explained by hydrogen-deuterium exchange processes and was confirmed by electrochemical oxidation of D4-APAP in a deuterated water/acetonitrile solvent (see Section SI-4 for the detected mass voltammogram). Here, two hydrogen−deuterium exchanges were observed, which probably took place at the position of the hydrogens, which are bonded to oxygen or nitrogen, respectively. To conclude, the system phenol/13C6-phenol seems to be better suited for labeling of small peptides compared to APAP/D4-APAP as fully resolved signals for differentially labeled GSH could be obtained and no hydrogendeuterium exchanges can occur, which would result in a more complex mass spectrum. Protein Labeling with Electrochemically Generated Reactive Intermediates. Since differential labeling of the tripeptide GSH could successfully be performed, in further experiments, the reactivity of the generated intermediates benzoquinone and NAPQI toward larger molecules like proteins was tested. Therefore, each compound (phenol, 13 C6-phenol, APAP, and D4-APAP) was electrochemically oxidized at a constant potential and allowed to react with the respective protein. The instrumental setup applied is schematically shown in Figure 1c. Instead of GSH, a protein solution was added to the effluent of the cell and prior to MS detection, a LC separation was integrated into the system. Thus, salts and the excess of labeling compounds were separated from the protein fraction in order to ensure a sufficient signal-to-noise ratio for the proteins in the ESI-MS measurements and to exclude noncovalent clusters of reactive intermediates and proteins. In the first experiments, labeling of the whey protein βlactoglobulin A (LGA; 18 363 Da) was investigated. LGA is relatively small in size, structurally homogeneous, and therefore a suitable model protein. It consists of 162 amino acids including five cysteine moieties. Four of them are bound in disulfide bonds and one is free and, thus, directly accessible for modification with electrophiles. In Figure 4a−d, the deconvoluted mass spectra of LGA and the corresponding adducts after incubation with the oxidized labeling compounds are shown. Labeling with benzoquinones Q and 13C6-Q, respectively, enables quantitative reaction of the free cysteine moiety of LGA. In both cases, only one adduct was formed and the signal originating from the native protein could not be detected after labeling. The respective mass differences of the native LGA and the LGA adducts confirm Q (18 471 Da; ΔM = 108 Da) and 13 C6-Q (18 477 Da; ΔM = 114 Da) to be the reactive species. In contrast, incubation of LGA with electrochemically oxidized APAP or D4-APAP resulted in more complex mass spectra. Although labeling could again be performed quantitatively, a distribution of adducts with various reactive intermediates was obtained. The main signal was identified to be the adduct of LGA with NAPQI (18 513 Da; ΔM = 150 Da) and D4-NAPQI (18 516 Da; ΔM = 153 Da), respectively. Moreover, two additional signals were successfully identified. On the one hand, it could be shown that dehydrogenated dimers of APAP and D4-APAP were formed upon electrochemical oxidation. As

Figure 4. Deconvoluted mass spectra obtained after adduct formation of β-lactoglobulin A (LGA; 18 363 Da) with (a) benzoquinone (18 471 Da), (b) 13C6-benzoquinone (18 477 Da), (c) benzoquinone (18 470 Da), NAPQI (18 513 Da), and APAP dimer (18 662 Da), and (d) D4-benzoquinone (18 473 Da), D4-NAPQI (18 516 Da), and D4APAP dimer (18 667 Da).

these species are also unsaturated electrophiles, modification of thiol groups was observed in both cases (dimer of APAP: 18 662 Da, ΔM = 299 Da; dimer of D4-APAP: 18 667 Da, ΔM = 304 Da). On the other hand, LGA adducts of the corresponding benzoquinones Q (18 470 Da; ΔM = 107 Da) and D4-Q (18 473 Da; ΔM = 110 Da), which were formed upon hydrolysis and deamination of NAPQI, could be E


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Analytical Chemistry identified. Due to the complexity of the obtained mass spectra after protein modification and the previously discussed H/Dexchange processes, it can be concluded that the system consisting of phenol and 13C6-phenol is much better suited than acetaminophen and D4-acetaminophen for protein labeling experiments. In order to demonstrate the broad applicability of the developed labeling strategy, larger proteins of higher complexity were investigated. Human serum albumin (HSA) is a heterogeneous protein and is present in two isoforms: mercaptalbumin (HMA, 66 438 Da) contains one free cysteine, whereas in nonmercaptalbumin (HNA, 66 558 Da), the cysteine moiety is blocked via a post translational disulfide formation with small thiol containing biomolecules like cysteine or GSH. For electrochemically oxidized phenol and 13C6phenol as well as for APAP and D4-APAP, quantitative labeling of HMA was successfully performed (see Section SI-5 for the detected and deconvoluted mass spectra). In all cases, after derivatization, no signal for the HMA isoform was detected anymore, thus indicating that the cysteine residue was labeled quantitatively. Comparable results were obtained for bovine serum albumin (BSA), which also occurs in two isoforms, one of which contains a free cysteine. The corresponding cysteine containing isoform was again labeled quantitatively with reactive benzoquinone and NAPQI intermediates (see Section SI-6 for the detected and deconvoluted mass spectra). The red blood cell protein hemoglobin (Hb) is a tetramer consisting of two α-chains (α-Hb) and two β-chains (β-Hb). While α-Hb has one free cysteine, β-Hb contains two. Labeling with Q gives the labeled α-Hb and 2-fold labeled β-Hb, whereas with 13C6-Q, αHb, and β-Hb were labeled only once. Furthermore, derivatization with NAPQI and D4-NAPQI results in the formation of α-Hb adducts only (for detected and deconvoluted mass spectra, see Section SI-7). To conclude, quantitative protein labeling can easily be achieved by reacting cysteine containing proteins with electrochemically oxidized phenol and 13 C6-phenol. In comparison, labeling with reactive metabolites of APAP and D4-APAP is not always quantitative and, in addition, mass spectra are more complex. Another protein, which was extensively investigated, is human carbonic anhydrase I (CAI). The metalloprotein CAI, which uses Zn2+ ions as cofactor, is highly abundant in red blood cells, catalyzes the reaction between carbon dioxide and water, and plays an important role in the CO2-transport in the human body. CAI contains 261 amino acids including one free cysteine moiety and has a mass of 28 781 Da. The obtained mass differences of ΔM = 108 Da (28 889 Da) and ΔM = 114 Da (28 895 Da) after incubation of CAI with electrochemically treated phenol and 13C6-phenol indicate that the free cysteine residue was quantitatively labeled with Q and 13C6-Q, respectively (see Figure 5). However, when differential labeling of CAI with a mixture of Q and 13C6-Q was performed, only one signal for a protein adduct can be detected (see Section SI8 for the deconvoluted mass spectrum). Since the deconvoluted mass of the detected CAI adducts (28 892 Da) is between the masses of the CAI adduct with Q and 13C6-Q, respectively, it can be concluded that CAI was successfully labeled. Nevertheless, the obtained mass difference of six mass units is not sufficient to detect resolved signals from the differentially labeled proteins with this mass spectrometric setup. Therefore, the corresponding proteins have to be cleaved into smaller peptides in order to enable the detection of the mass difference on the peptide level.

Figure 5. Deconvoluted mass spectra of unmodified human carbonic anhydrase I (CAI; 28 781 Da) and quantitatively labeled CAI with benzoquinone (CAI + Q; 28 889 Da) and 13C6-benzoquinone (CAI + 13 C6-Q; 28 895 Da).

Tryptic Digest of Differentially Labeled Proteins. In order to obtain fully resolved signals on the peptide level and to clearly define the thiol group to be the binding site of the generated species in CAI, a tryptic digest of differentially labeled CAI was carried out. The protein was cleaved into smaller peptides, which allows the LC separation and determination of the exact masses of the tryptic peptides by means of high-resolution MS. In Figure 6a,b, the obtained LC/MS chromatograms are shown. Using a C5 reversed phase column (wide pore) and a binary gradient consisting of 0.1% formic acid and acetonitrile, most of the peptides were successfully separated and detected, although they strongly differ in their size (4−40 amino acids). The T19 peptide contains 40 amino acids, one of which is the free cysteine moiety of CAI and has a retention time of tR = 21.43 min (Figure 6a). The detected isotope pattern of the unmodified T19 peptide is shown in Figure 6c. In comparison, the retention time of the modified peptide is extended (tR = 24.72 min), as can be seen in the LC/MS chromatogram of labeled and digested CAI in Figure 6b. The retention time shift can be explained due to the additional nonpolar hydroquinone in the modified T19* peptide. On the basis of the exact mass, the T19 peptide can be identified to be the binding site of the reactive intermediates, thus indicating that the thiol group of free cysteine was successfully labeled. The obtained mass difference based on the heavy isotope labeling in 13C6-Q can be observed in the mass spectrum (Figure 6d). The charge state of 3+ results in a mass difference of Δm/z 2 and allows the unambiguous identification of the differentially labeled peptide. Moreover, these experiments were additionally carried out with differentially labeled hemoglobin. After tryptic digestion, the differentially labeled peptide from the α-chain as well as one modified peptide from the β-chain could easily be identified on the basis of the obtained mass difference (for LC/MS chromatograms and mass spectra of the differentially labeled peptides, see Section SI-9). Therefore, these results underline the great potential of the developed method for the identification of cysteine containing peptides in order to obtain information on the cysteine content in a protein.

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CONCLUSION Electrochemical oxidation of phenol, 13C6-phenol, acetaminophen, and D4-acetaminophen leads to the generation of reactive intermediates, which have shown a specific reactivity toward nucleophilic thiol groups in cysteine containing biomolecules. F


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Figure 6. LC/MS chromatograms of the obtained tryptic peptides of (a) unmodified CAI and (b) differentially labeled CAI. Detected mass spectra of the (c) unmodified T19 and (d) differentially labeled T19* peptide. The mass difference of Δm/z 2 of the labeled T19* can be traced back to the heavy isotope labeling (13C6-Q) in the charge state 3+.

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The tripeptide glutathione could successfully be labeled with electrochemically generated benzoquinone and 13C6-benzoquinone or NAPQI and D4-NAPQI, respectively. Differentially modified glutathione resulted in a mass difference based on the corresponding heavy isotope labeled compound. Furthermore, the generation of mass voltammograms of the glutathione adducts allowed the determination of the optimum oxidation potential for the generation of reactive intermediates. Afterward, the optimum potential was applied for the generation of reactive species in order to achieve quantitative protein labeling. Therefore, the proteins β-lactoglobulin A, human serum albumin, bovine serum albumin, hemoglobin, and human carbonic anhydrase I were investigated. While for phenol and 13 C6-phenol the quantitative conversion of the free cysteines with only one reactive intermediate was obtained, acetaminophen and D4-acetaminophen labeling was not quantitative for all proteins. Furthermore, more than one reactive intermediate was formed, and hydrogen-deuterium exchange processes were observed. Therefore, protein labeling with phenol/13C6-phenol has a higher capability for differential labeling compared to acetaminophen/D4-acetaminophen. Tryptic digestion of carbonic anhydrase I, which was differentially labeled with benzoquinone and 13C6-benzoquinone, allowed the unambiguous determination of cysteine to be the binding site of the reactive intermediates in the protein. Moreover, the introduced mass difference can be detected at the peptide level and thus allows a simplified identification of cysteine containing tryptic peptides in the LC/MS data. To summarize, the developed method for differential and quantitative protein labeling is of great potential for proteomics experiments as information on the cysteine content in proteins can easily be obtained. Hence, additional information for protein identification can be provided and further be used for database searches.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02497. Settings and operating conditions of the mass spectrometers; tryptic digest of hemoglobin adducts; electrochemical oxidation and GSH adduct formation; mass voltammogram of D4-APAP in deuterated solvents; labeling of human serum albumin; labeling of bovine serum albumin; labeling of human hemoglobin; differentially labeled carbonic anhydrase I; tryptic digest of unmodified and differentially labeled hemoglobin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +49 251 83-33141. Fax: +49 251 83-36013. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the NRW Graduate School of Chemistry for financial support in the form of a Ph.D. scholarship for L.B.

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REFERENCES

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