Electrocatalytic Assay for Monitoring Methylglyoxal-Mediated Protein

Dec 25, 2014 - Protein structural transition at negatively charged electrode surfaces. Effects of temperature and current density. Hana Černocká , V...
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Electrocatalytic Assay for Monitoring Methylglyoxal-Mediated Protein Glycation Marika Havlikova,† Martina Zatloukalova,† Jitka Ulrichova,† Petr Dobes,‡ and Jan Vacek*,† †

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic ‡ Regional Centre for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic S Supporting Information *

ABSTRACT: Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around −1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

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dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. Methylglyoxal irreversibly reacts with amino groups in lipids, nucleic acids, and proteins, forming methylglyoxalderived advanced glycation end-products (MAGEs).8 Many laboratories have developed various experimental techniques and approaches for protein glycation analysis and the long-term monitoring of glycation processes leading to various glycation products, especially AGEs as shown in Table S1 (Supporting Information). Accordingly, we can consider the following methods proposed for the analysis of frequently studied glycation models, which are primarily albumins (reviewed in refs 7 and 9). Thiobarbituric acid,10 nitroblue tetrazolium,11 and phenylhydrazine12 assays are common colorimetric methods. Glycated albumins can be analyzed by enzymatic methods using ketoamine oxidase and bromocresol purple.13 As for separation approaches, boronate affinity chromatography,14 furosine procedure-HPLC,15 and phenylboronate-based electrophoresis16 were developed. Phenylboronate can also be used in an immunoassay called enzyme-

rotein glycation is the result of the covalent bonding of the protein molecule with sugars and their metabolic byproducts via a nonenzymatic process. Thus, the glycation reaction is sometimes also referred to as “nonenzymatic glycosylation”, which may occur either inside an organism, “endogenous glycation”, or outside the body, “exogenous glycation”, when sugars are cooked with proteins. The glycation of proteins occurs by a complex series of sequential and parallel reactions that form a Schiff’s base, Amadori products, and advanced glycation end-products (AGEs).1 Many different adducts may be formed,2,3 and some of them are colored and fluorescent in nature. Glycation is a process with high biological relevance that impairs the function of proteins and thus participates in the regulation of many physiological functions and has various clinical consequences.2−7 In the physiological setting, glucose and other saccharides are important glycation agents, but the most reactive glycation agents are the α-oxoaldehydes, glyoxal, methylglyoxal, and 3‑deoxyglucosone. Methylglyoxal is the most significant glycation agent in vivo, being one of the most reactive dicarbonyl molecules in living cells. This compound is an unavoidable byproduct of glycolysis, arising from the nonenzymatic β‑elimination reaction of the phosphate group of © 2014 American Chemical Society

Received: September 30, 2014 Accepted: December 25, 2014 Published: December 25, 2014 1757

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chemical methods are given in the figure legends. Every sample III was dialyzed (see previous paragraph) for electrochemical monitoring purposes. Native PAGE. Native PAGE was carried out on 10% polyacrylamide gels (Mini PROTEAN Tetra Cell Systems, BioRad, Hercules, CA). In total, 10 μg of protein samples I, II, and III were loaded without denaturation. After separation at pH 8.8, protein bands were visualized by staining with Coomassie Brilliant Blue. Changes in the migration positions of polypeptide bands were evaluated as described previously.21 Fluorescence Spectroscopy. The fluorescence intensities of 15-fold diluted samples (I, II, and III) in 0.1 M PBS (pH 7.4) were measured at 420 nm after excitation at 350 nm, using a fluorescence microplate reader (Infinite M200 PRO, Tecan) operating at room temperature; integration time 20 μs; excitation bandwidth 9 nm; emission bandwidth 20 nm. Other details were as reported previously.23 2D-Isoelectric Focusing SDS-PAGE (IEF/SDS-PAGE). IEF was performed on 7 cm, pH 3−10 linear immobilized pH gradient (IPG) strips (BioRad, Hercules, CA). The strips were rehydrated overnight in a solution containing 5 M urea, 2 M thiourea, 2% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 20 mM DTT (dithiothreitol), 5 mM TCEP (tris(2-carboxyethyl)phosphine), 0.5% ampholytes 4-7, 0.25% ampholytes 3-10, and 2% IPG buffer pH 3−10 containing 10 μg of the protein sample. Strips were covered with mineral oil. Focalization was carried out at 8500 Vh, with a maximum of 2500 V at 20 °C, using a Protean IEF Cell (BioRad, Hercules, CA). Prior to the second dimension (SDS-PAGE), strips were incubated in the equilibration buffer with 1% DTT, 6 M urea, 4% SDS, 30% glycerol, 50 mM Tris/ HCl (pH 8.8), 0.5% bromphenol blue for 15 min, then with the same buffer but with 4% iodoacetamide instead of DTT for the next 15 min. SDS-PAGE was carried out on 10% polyacrylamide gels. Polypeptide spots were stained with Coomassie Brilliant Blue.22 Molecular Model of BSA. The BSA monomer model (PDB code 3F4S) was visualized in PyMOL (The PyMOL Molecular Graphics System, version 1.5.0.4; Schrödinger, LLC). Primary sequence (Cys, C; Lys, K; Arg, R; His, H; electrocatalytically active amino acid residues are highlighted):

linked boronate immunoassay (ELBIA),17 an immunoassay without the use of phenylboronate has been developed for albumin-AGE detection.18 As for electrochemical methods, the “indirect” analysis of albumin and hemoglobin glycation based on oxygen reduction at a pyrolytic graphite electrode has been reported.19 To the best of our knowledge, a “direct” and sensitive electrochemical method focused on the observation of protein electroactivity or the electroactivity of formed glycation products (adducts) has not been reported to date. Here we present a novel electrochemical label-free method for the in vitro monitoring of protein glycation via observing changes in intrinsic electroactivity of the protein. The study covers bovine and human serum albumins (BSA and HSA) and lysozyme (LYZ) as model protein molecules and methylglyoxal20,21 as the glycation agent. The electrochemical results presented here are supported by previously developed complementary methods, i.e., native (PAGE) electrophoretic assay,21 2D isoelectric focusing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),22 and the fluorescence spectroscopy method.23 Negative control experiments were performed using the AGE formation inhibitor aminoguanidine (pimagedine) as previously proposed.24

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EXPERIMENTAL SECTION Chemicals. Proteins and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), BioRad Laboratories (Hercules, CA), or GE Healthcare Life Science. All solutions were prepared using reverse-osmosis deionized water (Ultrapur, Watrex, CZ). The sample preparation and analyses were performed under aerobic conditions. The pH-values of the buffer solutions were measured with a pH/ORP meter (HI2211) equipped with an HT 1131 electrode (HANNA Instruments). Sample Preparation. Samples were prepared as follows: sample I (10 mg/mL BSA, HSA, or LYZ in 0.1 M PBS, pH 7.4), sample II (sample I + 50 mM methylglyoxal), and sample III (sample II + 10 mM aminoguanidine) were incubated at 37 °C for 72 h. All samples were then centrifuged at 10 000g for 10 min at 4 °C and the supernatants were analyzed. Where needed, supernatants of the samples were collected and dialyzed overnight using a D-Tube Dialyzer Mini, MWCO 12−14 kDa (Novagen, Podenzano, Italy) against deoinized water at 4 °C prior to analysis. Protein content was determined by Bradford assay in 15-fold diluted samples.25 In experiments demonstrating a gradual suppression of glycation, sample III contained various concentrations of aminoguanidine (2.5, 5, 7.5, or 10 mM). For better orientation, see Scheme S1 in the Supporting Information. Electrochemical Measurement. The samples (I, II, and III) were analyzed using out-of-phase alternating-current voltammetry (ACV) and constant-current stripping chronopotentiometric analysis (CPSA). Two types of working electrodes were used: a HMDE (hanging mercury drop electrode; area, 0.4 mm2) and a silver solid amalgam electrode, AgSAE (area, 0.8 mm2).26 Before the measurement, samples were diluted to a final 500 nM (for HMDE) or 250 nM (for AgSAE) protein concentration in an electrochemical cell containing 5 mL of the supporting electrolyte (Britton-Robinson buffer, pH 6.5). All measurements were performed at room temperature with a μAutolab III analyzer (EcoChemie, NL) connected to a VAStand 663 (Metrohm, Herisau, Switzerland) in a threeelectrode setup with Ag/AgCl3 M KCl and Pt-wire as reference and auxiliary electrodes, respectively. Settings for electro-

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RESULTS AND DISCUSSION Experimental Design and Principle of the Method. The main goal of this study is to develop a novel electrochemical assay for monitoring glycation processes in proteins. The experimental design of the study is based on the investigation of three different samples where proteins (10 mg/mL) were incubated in 0.1 M PBS (pH 7.4) at 37 °C for 3 days in all experiments. Sample I represents the native 1758

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protein, while in sample II the protein was incubated with 50 mM methylglyoxal glycation agent,20,21 and sample III was the same as sample II but with the addition of aminoguanidine (10 mM, if not stated otherwise), which is a glycation inhibitor24 (Scheme S1 in the Supporting Information). Bovine serum albumin (BSA) was primarily used for the development and optimization of the method and later tests used for other proteins frequently used in protein glycation research, i.e., human serum albumin (HSA) and lysozyme (LYZ).7,27 After completing the incubation period (1, 2, or 3 d) the protein samples were centrifuged, the total protein content was measured by the Bradford method25 in supernatants, and the supernatant low-molecular-weight (LMW) soluble protein fraction was diluted to a final concentration of 250 or 500 nM directly in the electrochemical cell. Britton-Robinson buffer (pH 6.5) was used as the supporting electrolyte and measurement was done using CPSA in connection with two types of Hg-electrodes, a HMDE and AgSAE. The general principle of the proposed assay is to monitor an electrocatalytic process called “catalytic hydrogen evolution reaction” (CHER), where the protein serves as the catalyst.28−30 The result of the measurement is an electrocatalytic CPS signal observable at negative potentials at Hgelectrodes known as peak H.26,27 A range of proteins have been studied using CPS peak H (for BSA see refs 31−33), and it was shown that its height, potential, and shape is not only dependent on the protein concentration in the sample but also on the protein’s structural changes and its intermolecular interactions with several ligands. Concretely, proton-donating amino acid (aa) residues, Cys and the basic aa residues Lys, Arg, and His,29,30,33 are responsible for the electrocatalytic process, i.e., CHER. One of the driving factors for CHER is the accessibility of electrocatalytically active aa residues to the surface of the working electrode. In this respect, it is evident that the proton-donating aa residues involved in the CHER have to be located at the protein surface.34,35 The total distribution of Cys, Lys, Arg, and His residues in the BSA molecule is shown in Scheme 1A. For comparison, those same aa residues which are at least 10% accessible to the solvent are highlighted using the surface molecular model (Scheme 1B). The aa residues highlighted are most likely involved in the CHER. In more detail, the principle of our approach is connected to the covalent modification (glycation) of the above-mentioned aa residues because the submolecular targets for nonenzymatic protein glycation are primarily Lys, Arg, Cys, and to a limited extent also His.7 Thus, if the glycation reaction proceeds, the electrocatalytically active aa residues are not able to contribute to the CHER, which is reflected in the changes in peak H in the investigated protein samples. In albumins, Lys and Arg and only one Cys (Cys-34) are involved in the glycation process, because other Cys residues participate in the formation of disulfide bonds.7 As for His residues, to our knowledge they probably only undergo partial glycation, but it must be noted that our knowledge in this area is incomplete.7 The number and precise enumeration of aa residues exposed to the BSA surface can be found in Table S2 (see the Supporting Information). Taking into account the fact that the glycation targets in proteins are the same aa residues as those participating in the electrocatalytic reaction, we are able to selectively monitor glycation processes via the decrease in CPS peak H. Electrocatalytic Analysis of BSA Glycation. First, we analyzed the glycation of BSA according to the experimental

Scheme 1. Molecular Structure of BSA (PDB Code 4F5S, Monomer A) with Electrocatalytically Active Cys (Cyan), Lys (Magenta), His (Orange), and Arg (Brown) Residues Highlighteda

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(A) The structure is displayed in cartoon representation and colored according to secondary structure elements: α-helices in red, β-sheets in yellow, and coil structures in green. The aa residues are highlighted as spheres. (B) Electrocatalytically active aa residues exposed to the surface (with solvent accessibility of at least 10%) of BSA are highlighted. Left (front) and right (back) images of the same molecule are mutually rotated by 180° along the vertical axis for both panels A and B.

design described above, see Scheme S1 in the Supporting Information. Under the given experimental conditions using HMDE, native BSA (sample I) gives a CPS peak H at a potential of around −1.85 V (Figure 1). A similar signal was observed using the AgSAE at a less negative potential of −1.70 V (Figure 1, inset). With increasing incubation time of

Figure 1. CPS records of native (sample I) and glycated (sample II) BSA after 1, 2, and 3 days of incubation with 50 mM methylglyoxal using HMD and AgSA (in inset) electrodes. CPSA measurement: concentration of BSA (500 nM for HMDE and 250 nM for AgSAE experiments), accumulation time (60 s only for AgSAE experiments), stripping current (−35 μA for HMDE and −30 μA for AgSAE experiments), and supporting electrolyte (Britton-Robinson buffer, pH 6.5). Glycation experiment: incubation was carried out with 10 mg/mL BSA + 50 mM methylglyoxal in 0.1 M PBS (pH 7.4) at 37 °C. 1759

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protein in the electrochemical cell for CPSA. Theoretically, it is possible to extend the incubation period for BSA with glycation agents to up to 21 days, which is the time required for albumin elimination under physiological conditions;7 however, experiments lasting 3−5 days are preferred by most authors. In contrast to the above, a short-term glycation process can also be performed for the analysis of early glycation products,5 which is shown for BSA glycated for 6 h using 50 mM methylgyloxal in Figure S1A (see the Supporting Information). These results indicated that CPS analysis can be utilized not only for longterm glycation monitoring but also a relatively small amount of glycated protein can be identified in the excess of native BSA in the sample. Another important aspect in our approach could be connected to the adsorption behavior of the investigated proteins and the surface coverage of the working electrodes in the presence of the protein samples. To elucidate adsorption/ desorption phenomena, samples I and II incubated for 3 days were analyzed by out-of-phase ac voltammetry (ACV) at HMDE according to the previously reported methodology.35 ACV records for sample I were in good agreement with previously published data for native BSA,35 and these records were practically the same as the ac voltammograms of sample II. The surface of the working electrodes was fully covered with the tested proteins at a concentration of 500 nM for both HMD and AgSA electrodes under the experimental conditions used (see Figure 1). The calibration curves for native BSA were performed with a preconcentration step (time of accumulation, tA = 15 s for HMDE and 60 s for AgSAE) in the concentration range 50− 500 nM using a HMDE (R2 = 0.9952, y = 0.303x − 23.274 (s V−1/10−9 M)) and AgSAE (R2 = 0.9983, y = 10.976x − 13.76 (s V−1/10−9 M)). Using optimized conditions for ex situ measurement (for experimental details see ref 38) and tA = 120 s, the limits of detection (LODs = 3 S/N) for BSA glycated for 1 day were found to be 500 pM and 10 nM for HMDE and AgSAE, respectively. The samples were immobilized onto the working electrode’s freshly renewed surface in 5 μL volumes that correspond to LOD = 166 pg of glycated BSA per analyzed sample if the HMDE was used. The quantitative analysis results performed with a 1-d glycated BSA sample are summarized in Figure S1B in the Supporting Information. The sensitivity of CPSA for the detection of glycated BSA is significantly higher than other methods frequently used in protein glycation research. For an overview of the methods applied for BSA glycation analysis, see Table S1 in the Supporting Information. Neither of the methods reviewed in Table S1 in the Supporting Information gives a better LOD than CPSA, and it is fair to say that primarily (radio)immunochemical approaches provide a comparable sensitivity to our methodology (Table S1 in the Supporting Information). It is important to note that comparing the selected assays is quite controversial because the published studies used different sample pretreatment procedures and their expression of LODs was also not identical. In terms of electrochemical methods, the proposed electrocatalytic assay is many times as sensitive as the recently published electrochemical approach by Yang et al.,19 where glycated hemoglobin and HSA solubilized in DMSO were immobilized onto pyrolytic graphite electrode at concentrations 10 and 40 mg/mL, respectively. Complementary Methods and Inhibition of Glycation. In the next step we compared the results acquired by electrocatalytic assay with the results of complementary

BSA with the glycation agent (sample II), a decrease in CPS peak H height was observed with complete signal disappearance after 2−3 days of incubation, as determined using both electrodes (Figure 2A). The AgSAE was used to improve the

Figure 2. Time dependence of CPS peak H height on glycation of BSA, without (A) and with (B) a dialysis step prior to CPS analysis. Native BSA, sample I (□); glycated BSA, sample II (■). Protein content in LMW fractions of native (○) and glycated (⧫) BSA (dotted gray lines for both) measured by the Bradford assay is shown on the right Y axis. Data are means ± SD of six measurements at HMDE. Error bars smaller than the plotted symbols are not visible. Native protein = 100% = 32.3 s V−1 (peak H height, CPSA) and 0.6 AU (absorbance at 595 nm, Bradford assay).

applicability of the electrocatalytic assay, especially for the development of novel electrochemical sensors and use in continual flow detection platforms, where a HMDE is not fully applicable.36 The effect of competing substances on the electrocatalytic signal of BSA was evaluated in the next part of the experimental work. In this sense, we examined the effect of methylglyoxal in sample II because the glycation agent is present at the relatively high concentration of 50 mM. For this reason we included the dialyzation step for removing methylglyoxal prior to electrochemical analysis (Figure 2B). As a result, CPS signals were fully comparable between the nondialyzed and dialyzed samples (compare parts A vs B in Figure 2) indicating that the presence of methylglyoxal did not contribute to false positive or negative results. An important factor which has to be taken into account is the time period for sample glycation. The incubation was set up to last 3 days, since it is well-known that proteins undergo aggregation and similar processes during long-term protein incubation.37 The proteins were subjected to centrifugation as stated above (Scheme S1 in the Supporting Information) to eliminate the insoluble high-molecular weight (HMW) fraction. After centrifugation, the concentration of protein in the soluble LMW fraction in the supernatants was verified by total protein analysis by the Bradford assay (Figure 2, right Y axis) and corrected to the required 250 or 500 nM final concentration of 1760

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methods frequently used for nonenzymatic glycation analysis.21−23 In addition to samples I and II, sample III containing 10 mM aminoguanidine glycation suppressor24 was first analyzed by native PAGE (Scheme S1 in the Supporting Information). The resulting electrophoretograms (Figure 3A)

Figure 3. Effect of glycation on electrophoretic mobility of BSA by native PAGE. (A) Electrophoretograms of sample I (native BSA), sample II (glycated BSA), and sample III (glycation suppressed). (B) Time-dependent effect of 10 mM aminoguanidine on electrophoretic mobility of BSA. Glycated BSA (■), glycation suppressed with 10 mM aminoguanidine (□). Data are normalized to the mobility of native BSA (sample I = 100%). Data are means ± SD of three measurements. Error bars smaller than the plotted symbols are not visible.

show an increase in the electrophoretic mobility of glycated BSA (sample II) in comparison to the native protein (sample I). This increase in mobility is related to protein charge changes after glycation because positively charged aa residues (Lys and Arg) are involved in condensation reactions with the glycation agent.39 This leads to the depletion of positive charge in glycated proteins and results in higher protein attraction to the anode in the electrophoretic bath. When the glycation inhibitor was present in samples (i.e., samples III) the glycation process was suppressed and no increase in protein electrophoretic mobility was observed, which was well documented after measuring the migration zones for single electrophoretograms at various incubation time intervals (Figure 3B). The changes in protein charge, observed after glycation, are simultaneously reflected in changes in protein pI values, which was confirmed by another complementary method, i.e., 2D electrophoresis with a pH gradient of 3−10 (Figure 4). The 2D-electrophoretic patterns for glycated samples were in good agreement with the electrophoretic patterns reported by other authors.22 After the evaluation of electrophoretic experiments, a detailed comparative study covering fluorescence spectroscopy was conducted23 in the presence or absence of aminoguanidine, a glycation inhibitor, and consequently compared with the results of electrochemical analysis. The fluorescence signal (ex/ em wavelengths 350/420 nm) increased during glycation in sample II in comparison to the fluorescence signal of native BSA (Figure 5A). In addition to this, for sample III, where glycation was suppressed, the fluorescence signals of BSA were the same as for sample I. The increase in fluorescence is related to the formation of glycation products (i.e., MAGEs, see the Introduction), which are conjugated systems (abundant in double bonds) and thus actively fluorescent.23 In the next part of the experimental work, we focused on monitoring the effects of different concentrations of aminoguanidine (2.5−10 mM). The samples were analyzed after 3 days of incubation and were dialyzed for 24 h prior to CPS analysis (for details, see the Experimental Section). Dialysis is important for eliminating interfering species linked to the

Figure 4. 2-D isoelectric focusing SDS-PAGE electrophoretograms of native BSA (A), glycated BSA (B), and BSA where glycation was suppressed with 10 mM aminoguanidine (C). Glycation experiment: incubation was carried out with 10 mg/mL BSA + 50 mM methylglyoxal in 0.1 M PBS (pH 7.4) at 37 °C for 3 days. For comparison, a separation of unglycated and glycated BSA samples by native PAGE is documented in Figure 3A.

application of glycation inhibitors and other possible additives in the samples. With increasing concentration of aminoguanidine, a proportional decrease in fluorescence signals and increase in the electrocatalytic response of BSA were observed (Figure 5B). This result indicated that our method is not only selective for the analysis of glycation processes but can also be used for monitoring glycation inhibition effects. Finally, the applicability of the electrocatalytic assay was tested by using other protein models (HSA and LYZ) and the analytical outputs were compared with the BSA results described above. We analyzed samples I, II, and III for each protein separately after 3 days of incubation using the AgSAE (Figure 6). Under identical experimental conditions, the same effects observed for BSA were acquired with HSA and LYZ. Electrocatalytic peak H was significantly higher for LYZ than for BSA and HSA (compare parts A and B vs part C in Figure 6) with respect to the structural diversity and high amount of basic aa residues in LYZ, which fully corresponds to its pI of 11.35.40 For comparison, the pI-value of albumins is ∼4.7. The same results as shown in Figure 6 were also obtained with a HMDE (not shown). The electrochemical response of proteins due to the CHER provides practically all the proteins investigated to date (reviewed in ref 28), showing that CHER is a universal phenomenon. This work demonstrated the applicability of the electrochemical approach based on a CHER for BSA, HSA, and LYZ, indicating a nonproblematic transfer of 1761

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function investigation and the detection of protein intermolecular interactions.43,44 Here, a label-free electrocatalytic assay for monitoring nonenzymatic protein glycation with methylglyoxal as the glycation agent is reported. One of the main advantages is that the assay primarily follows protein surface modifications, in contrast to the other robust methods used for protein glycation analysis, i.e., fluorimetry and mass spectrometry. Using these methods, there is no way to monitor glycation processes that only involve surface-exposed aa residues in the analyzed proteins. Conventional optical spectroscopy and MS provide analytical signals indicating overall glycation without discriminating between processes localized at the protein surface vs processes occurring in the inner parts of the protein molecule. Thus, our approach primarily reflects “protein surface chemistry” in comparison to other methodologies. In addition to this, the methodology proposed could be the starting point for the further development of novel techniques for protein glycation detection, especially electrochemical sensors and versatile amalgam electrode-based sensing platforms.35,36

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Figure 5. (A) Time-dependent effect of glycation on fluorescence intensity of BSA at ex/em wavelengths 350/420 nm: glycated BSA (■) and glycation suppressed with 10 mM aminoguanidine (□). (B) Dose-dependent effect of aminoguanidine on fluorescence intensity (□) and CPS response (○) of BSA. Data are means ± SD of three measurements. Error bars smaller than the plotted symbols are not visible. Native protein = 100% = 140 au (emission at 420 nm, fluorimetry) and 32.1 s V−1 (peak H height, CPSA).

ASSOCIATED CONTENT

* Supporting Information S

[Scheme S1, graphical representation of sample handling and analytical methods used for detecting protein glycation; Figure S1, details on early glycation product detection and quantitative analysis of BSA; Table S1, overview of methods for BSA glycation analysis; Table S2, number of total and surfaceexposed Cys, Lys, Arg, and His residues in BSA molecule. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +420-585-632-303. Fax: +420-585-632-302. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are indebted to Dr. Nessar Ahmed (Manchester Metropolitan University, Manchester, U.K.), Prof. Dr. Emil Palecek, Dr. Veronika Ostatna, and Dr. Vlastimil Dorcak (Institute of Biophysics of the ASCR, Brno, CZ) for critical reading of the manuscript, to Dr. Bohdan Josypcuk for preparation of the AgSAE and to Mr. Ben Watson-Jones MEng for language correction. This work was supported by the Czech Science Foundation (Project No. 14-08032S, J.V.), by the Ministry of Education, Youth and Sports of the Czech Republic (MEYS)/COST Action EU-ROS, BM1203 (Project No. LD14033, J.V.), by the MEYS project No. CZ.1.07/2.3.00/ 30.0004, M.H, and by the European Regional Development Fund and the State Budget of the Czech Republic RECAMO CZ.1.05/2.1.00/03.0101, P.D.

Figure 6. CPS records (peak H) of bovine serum albumin, BSA (A); human serum albumin, HSA (B); and lysozyme, LYZ (C) at silver solid amalgam electrode (AgSAE). Native protein (sample I), glycated protein (sample II), and protein sample where glycation was suppressed with 10 mM aminoguanidine (sample III) for 3 days.

the developed assay for the glycation monitoring of a broad spectrum of other proteins. An interesting possibility for further studies is the fact that a similar electrocatalytic methodology reported here was recently shown to be applicable for the analysis of poorly water-soluble (transmembrane) proteins and lipoproteins.35,38,41 Thus, there is significant potential for the application of this methodology for monitoring the glycation of membrane proteins, which is still a current methodological problem.

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REFERENCES

(1) Ahmed, N. Diabetes Res. Clin. Pract. 2005, 67, 3−21. (2) Anguizola, J.; Matsuda, R.; Barnaby, O. S.; Hoy, K. S.; Wa, C.; DeBolt, E.; Koke, M.; Hage, D. S. Clin. Chim. Acta 2013, 425, 64−76. (3) Furusyo, N.; Hayashi, J. Biochim. Biophys. Acta 2013, 1830, 5509−5514. (4) Koga, M. Clin. Chim. Acta 2014, 433, 96−104. (5) Kulkarni, M. J.; Korwar, A. M.; Mary, S.; Bhonsle, H. S.; Giri, A. P. Proteomics Clin. Appl. 2013, 7, 155−170.

CONCLUSIONS Finding novel methods for investigating protein modifications is one of the most important aspects in the progress being made in protein science and proteomics today.42 Recently electrochemistry, with many modifications, was reported as a promising tool for protein trace analysis, protein structure and 1762

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DOI: 10.1021/ac503705d Anal. Chem. 2015, 87, 1757−1763