Article pubs.acs.org/JAFC
Nonenzymatic Modification of Ubiquitin under High-Pressure and -Temperature Treatment: Mass Spectrometric Studies Monika Kijewska, Karolina Radziszewska, Martyna Kielmas, Piotr Stefanowicz,* and Zbigniew Szewczuk Faculty of Chemistry, University of Wrocław, Wrocław, Poland ABSTRACT: The effect of high-pressure and/or high-temperature on the glycation of a model protein (ubiquitin) was investigated by mass spectrometry. This paper reports the impact of high pressure (up to 1200 MPa) on the modification of a ubiquitin using ESI-MS measurements. The application of glucose labeled with stable isotope allows a quantitative assessment of modification under the conditions of high-pressure (HPG) and high-temperature (HTG) glycation. A higher degree of modification was observed for the sample heated at 80 °C for 25 min under atmospheric pressure than for sample treated under high pressure. In samples treated at pressure below 400 MPa an insignificant increase of glycation level was observed, whereas high pressure (>600 MPa) has only a minor effect on the number of hexose moieties (Fru) attached to the lysine residue side chain. KEYWORDS: glycation, high-pressure glycation, Amadori products
■
INTRODUCTION High-pressure processing (HPP) preserves nutrients, natural flavor, and other sensory properties of food while extending the shelf life by preventing the growth of microorganisms by inducing structural changes to the cell membrane and inactivating important enzyme systems that disrupt metabolism of microorganisms.1,2 Some bacteria show resistance3 to pressures up to 800 MPa and or even higher. However, the application of high-pressure processing combined with increased temperature could be useful for sterilization of many products sensitive to high temperatures, such as food, cosmetics, and pharmaceuticals.4 HPP is a nonthermal preservation and pasteurization technique, which causes little or no changes in the sensory and nutritional attributes of the product being processed, unlike most conventional heat treatments.5 HPP accomplishes this by application of high hydrostatic pressures (between 100 and 1000 MPa) to food products. High pressure to about 3000 MPa does not affect covalent bonds in the molecules; therefore, many vitamins and provitamins as well as dyes, drugs; or other pharmacologically active molecules retain their unique characteristics.5,6 Bristow et al.7 and Moreno et al.8,9 reported the impact of high-pressure and alkaline conditions on the formation of Maillard products. The condensation of glyceraldehyde with diand tripeptides was hardly affected by a pressure increase (up to 500 MPa), and the reaction of glyceraldehyde, glycolaldehyde, or xylose with amino acids was slightly suppressed. For a glucose−lysine system, some stages of the Maillard reaction were affected differently.10 Staniszewska et al.11 proved that advanced glycation end products (AGEs) formed under high pressure (850 MPa) differ from those obtained thermally. In general, after high-pressure glycation (HPG), the products have a lower molecular mass than after high-temperature glycation (HTG). Immunoblotting experiments showed that the epitopes of the cross-linked glycation products formed in solution under high pressure are © 2014 American Chemical Society
not the same as those originating in dry conditions at high temperature. The antibodies against HTG products recognized HTG but not HPG products. This finding reveals that the biological properties of AGEs obtained by thermal treatment and high-pressure treatment may be different.11 The first investigations on the impact of pressure on modifications of proteinaceous amino acid residue side chains using glucose or more reactive dicarbonyl12 compounds revealed an increased level of pentosidine,13 whereas the observed formation of carboxyethylarginine from arginine did not occur at atmospheric pressure.14 In contrast, the generation of other Maillard reaction products, such as pyrraline, is hindered with the application of pressure.15 The effect of combined heat and pressure on the Maillard reaction between bovine serum albumin (BSA) and glucose was investigated by Buckow et al.16 The kinetic results showed that the protein−sugar conjugation rate increased with increasing temperature, but decreased with increasing pressure. However, five modified lysine residues were identified after 30 min of treatment at 200 MPa and 110 °C, that is, lysine residues K75, K204, K297/299, K548, and K557. Glycation of K548 was previously observed by Frolov et al.17 The effects of pressure and temperature on the reaction kinetics are antagonistic as an increase in pressure at constant temperature leads to a decrease in the entropy of the system.18 Considine et al.19 reported that BSA is relatively resistant to pressure denaturation, whereas it is easily denatured by heat and combined heat and high-pressure treatment. A similar investigation was performed on milk proteins in the absence and presence of different saccharides or dicarbonyl compounds.20 Without carbohydrates, protein cross-linking of casein is enhanced by pressure through the formation of dehydroalanine-derived lysinoalanine. In contrast, saccharide or Received: June 11, 2014 Accepted: December 18, 2014 Published: December 18, 2014 614
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 1. Deconvoluted ESI-MS spectra for ubiquitin glycated under high pressure in the range of 0.1−1200 MPa. High-Pressure Equipment. The high-pressure glycation experiments were carried out using a custom-made cylinder-piston type apparatus.28,29 The stoppered polypropylene syringe, containing the protein sample, was placed in a high-pressure vessel filled with hexane as a transmission medium and compressed (0.1−1200 MPa). High-Pressure Glycation. Ubiquitin (0.5 mg) and glucose (0.1 mg) were dissolved in 10 mmol/L aqueous ammonium formate (150 μL) at a 1:10 molar ratio and incubated at a given pressure (0.1−1200 MPa) for 24 h at room temperature (22 °C). After decompression, samples were diluted with 150 μL of a MeCN/H2O/HCOOH mixture (50:50:0.1 v/v/v). The samples were directly analyzed by ESI-MS. Additionally, the modified ubiquitin treated under a pressure of 800 MPa was hydrolyzed according to the procedure described below. High-Temperature Glycation. The high-temperature glycation of ubiquitin was performed according to Boratyński’s method.30,31 Briefly, ubiquitin and glucose or [13C6]glucose were dissolved in water at a 1:10 molar ratio and lyophilized. The dry lyophilisate was placed in an air oven at 80 °C for 25 min.27 The resulting preparation was lyophilized and directly used for MS experiments. Enzymatic Hydrolysis. Modified ubiquitin (1 mg) was dissolved in 5% aqueous formic acid (100 μL). Pepsin (10 μL of stock solution 1 mg/mL in water) was added, giving a 1:10 enzyme/substrate mass ratio. The reaction mixture was incubated for 5 h at 22 °C. The resulting digest was lyophilized and used for MS experiments. ESI-MS Measurement. ESI-MS experiments were performed using an Apex-Qe Ultra 7T instrument (Bruker Daltonic, Germany) equipped with an ESI source. The obtained fragments were subsequently analyzed by the ICR mass analyzer. The instrument was operated in the positive-ion mode and calibrated with the Tunemix mixture (Bruker Daltonic). The mass accuracy was better than 5 ppm. Spectra were recorded for 5 μmol/L protein in a MeCN/
dicarbonyl compound-induced cross-linking is reduced at high pressures. The authors hypothesized that protein cross-linking is decelerated due to either degradation of sugars or carbonyl compounds or formation of early Maillard products.20 Our recent work has been focused on the reaction of the lysine moiety with reducing sugars. The Amadori products, including glycoconjugates, are potentially useful markers of some diseases: diabetes, Alzheimer’s disease, or the aging process. We have reported an efficient method of synthesis,21 as well as selective mass spectrometric (ESI-MS) detection based on the neutral losses, borate complex formation,22,23 and isotopic (13C, 18O) labeling combined with enzymatic hydrolysis24−26 and sequencing of glycoconjugates using various methods of fragmentation (ECD, CID).27 The utilization of high pressure, an emerging technology in the area of food processing, has been increasing over the past two decades. Herein we report the impact of high pressure (up to 1200 MPa) on the level of glycation of a ubiquitin using ESIMS measurement. The quantification of the glycation level of ubiquitin was based on application of isotopic labeling [13C6].23
■
MATERIALS AND METHODS
Reagents. Reagents (including isotopically labeled [13C6]glucose; 99% 13C and pepsin) and solvents (including formic acid (HPLC grade), acetonitrile (LC-MS grade), and ammonium formate (LC-MS grade)) were purchased from Sigma. Ubiquitin was purchased from Aldrich and D-glucose from Eurochem. The reagents were used without further purification. 615
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 2. Deconvoluted ESI-MS spectrum of the mixture of ubiquitin modified thermally using [13C6]glucose and under high pressure (800 MPa) using standard glucose. H2O/HCOOH mixture (50:50:0.1 v/v/v). The sample was infused at a flow rate of 3 μL/min. The instrumental parameters were as follows: scan range, m/z 300−2500; drying gas, nitrogen; temperature of drying gas, 200 °C; potential between the spray needle and the orifice, 4.5 kV; source accumulation, 0.5 s; ion accumulation time, 0.5 s. The obtained mass spectra were analyzed using Bruker Data Analysis (Bruker Daltonic) software.
n is the number of Fru and In is the intensity of the ion. For protein samples incubated under pressure >600 MPa (Figure 1c−g), the signals corresponding to diglycated ubiquitin derivatives were observed. According to the literature, the critical pressure for denaturation of ubiquitin is approximately 600 MPa.32−34 This may explain the appearance of the doubly modified protein under higher pressure (600− 1200 MPa). The average level of modification was also calculated for 600 and 800 MPa pressures as 0.083 and 0.25, respectively. A further increase in pressure did not result in incorporation of another hexose moiety. The incubation of ubiquitin and glucose at 1000 MPa led to an additional modification, resulting in a 390.32 Da mass increase, which we were not able to identify accurately. In Figure 2, the ESI-MS spectrum of the mixture of ubiquitin modified thermally using [13C6]glucose and by a high-pressure (800 MPa) using standard glucose is presented. The signals corresponding to mono- and diglycated ubiquitin confirm the presence of protein modification resulting from the reaction with both labeled and unlabeled glucose. The same experiments were conducted to compare the level of glycation under atmospheric and high pressure (800 MPa), using regular and [13C6]glucose, respectively. In contrast to the results obtained for a high-temperature glycation using the same molar ratio of reagents as described previously,27 the application of a high pressure has minor influence on the degree of glycation. However, after the highpressure treatment, we observed additional signals in the MS spectra that were not observed after thermal treatment. We
■
RESULTS AND DISCUSSION High-Pressure Glycation. The effect of pressure on the Millard reaction between the model protein (ubiquitin) and a carbohydrate (glucose) was investigated. The series of experiments, including incubation of ubiquitin and glucose under high pressure in the range of 0.1−1200 MPa, were performed at isothermal conditions. The level of glycation was analyzed by ESI-MS. The deconvoluted mass spectra (Figure 1) reveal the signals corresponding to mono- and diglycated ubiquitin, although the spectra are dominated by the nonmodified protein. A control sample was prepared using the same molar ratio of reagents and incubated under atmospheric pressure. The peak corresponding to monoglycated ubiquitin was observed for pressures of 0.1 MPa (atmospheric) and 200 and 400 MPa (Figure 1a−c). The average level of glycation calculated according to eq 1 equals 0.08. The approximate level of glycation of a particular Lys residue was calculated by comparing intensities of ions of protein differing by the number of hexose moieties. n̅ =
∑ n*In ∑ In
(1) 616
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 3. ESI-MS spectrum of the mixture of hydrolysates of ubiquitin glycated thermally (HTG) and under pressure (HPG, 800 MPa).
lower degree of modification under high pressure in comparison to the thermal modification of ubiquitin at the same protein/sugar molar ratio, the number of detected modified peptides was lower because of reduced concentration of glycated ubiquitin. We tested the levels of glycation after the pressure and thermal treatment using isotopic labeling. High-temperature glycation was performed according to Boratyński’s method using [13C6]glucose, whereas ubiquitin glycated under a high pressure was prepared using a standard glucose. The obtained modified proteins were mixed at a 1:1 weight ratio and then hydrolyzed in the presence of pepsin. The mixture of peptic fragments was analyzed directly, without a chromatographic separation, on an Apex-Ultra ESI-MS spectrometer. The same procedure was used to compare the efficiency of the glycation under atmospheric (APG) and high pressure (800 MPa) (HPG) using ubiquitin and glucose and [13C6]glucose, respectively. The Amadori products obtained by the different treatments can be identified on the basis of mass difference caused by the use of [13C6] glucose. The search for glycated peptides can be performed automatically (in-house-designed spreadsheet) or by a visual inspection, recognizing the pairs of peaks with a defined m/z difference (m/z 6 for +1 ion, 3 Th for +2 ion, etc.). In the spectrum, signals corresponding to mixtures of hydrolysates of thermally and high-pressure glycated ubiquitin are presented. In the expanded range of this spectrum (Figure 3), a pair of signals corresponding to Amadori products 5−15 and 25−45 is shown. A significant difference in the signal intensity corresponding to
were not able to identify these byproducts. Further experiments are still needed to characterize these compounds and their mechanism of formation. The regioselectivity of glycation of lysine moieties in ubiquitin was evaluated by enzymatic hydrolysis of the modified protein (incubated under pressure of 800 MPa) by pepsin followed by a direct ESI-MS measurement. Pepsin is a nonspecific protease, with the preference for peptide bonds at the C-terminus from Phe and Leu.35 The crude hydrolysate was diluted with MeCN and then analyzed by ESI-MS. The glycation under high temperature and/or high pressure is relatively nonspecific. In the sequence of ubiquitin, there are seven lysine residues and the N-terminal amino group could be also glycated. Even though there are differences in the reactivity of the particular lysine moieties, every amino group may be glycated to some extent. Therefore, the sample of a glycated protein is an extremely complex mixture. In the analyzed hydrolysate, four peptides containing Amadori products, including residues 59−67, 5−15, 4−15, and 25−45, were found. The ECD fragmentation of modified peptide confirmed their sequence and the modification sites.27 The analysis of the spectra allowed for identification of the glycated lysine residues as Lys6, Lys11, Lys27, Lys29, Lys33, and Lys63 within the sequences of ubiquitin fragments. The modifications of Lys48 and the α-amino groups, which according to the literature27 are characterized by the lowest reactivity of all the potential glycation sites in ubiquitin, were not observed. In these studies, we received four identical peptide-derived Amadori products as in the case of thermally glycated ubiquitin.23,27 Due to a much 617
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Table 1. Identified Glycoconjugates in a Mixture of Hydrolysates of Ubiquitin Glycated Thermally Using Labeled Glucose ([13C6]Glucose) (HTG) and under High Pressure (800 MPa) Using Unlabeled Glucose (HPG) by MS m/z
a
glycated peptide
charge state
glycation methoda
found
calcd
intensity
modified Lys residue
4−15
1+
HPG glucose HTG [13C6]glucose
1483.864 1489.882
1483.862 1489.882
1.6 × 106 6.2 × 106
K6 or K11
3.88
5−15
1+
HPG glucose HTG [13C6]glucose
1336.794 1342.815
1336.793 1342.814
2.2 × 107 7.0 × 107
K6 or K11
3.18
25−45
2+
HPG glucose HTG [13C6]glucose
1300.210 1303.217
1300.208 1303.218
4.8 × 106 1.8 × 107
K27 or K29 or K33
3.75
59−67
1+
HPG glucose HTG [13C6]glucose
1257.623 1263.640
1257.621 1263.641
9.8 × 106 4.0 × 107
K63
4.08
intensity ratio IHTG/IHPG
HPG, glycation under high pressure; HTG, high-temperature glycation.
Table 2. Identified Glycoconjugates in a Mixture of Hydrolysates of Ubiquitin Incubated under Atmospheric Pressure Using Unlabeled Glucose (APG) and under High Pressure (800 MPa) Using Labeled Glucose ([13C6]Glucose) (HPG) by MS m/z
a
a
intensity ratio IHPG/IAPG
glycated peptide
charge state
glycation method
found
calcd
intensity
modified Lys residue
4−15
1+
APG glucose HPG [13C6]glucose
1483.863 1489.883
1483.862 1489.882
2.8 × 106 6.9 × 106
K6 or K11
2.45
5−15
1+
APG glucose HPG [13C6]glucose
1336.795 1342.814
1336.793 1342.814
3.7 × 107 7.5 × 107
K6 or K11
2.03
25−45
2+
APG glucose HPG [13C6]glucose
1300.210 1303.219
1300.208 1303.218
1.0 × 107 2.2 × 107
K27 or K29 or K33
2.2
59−67
1+
APG glucose HPG [13C6]glucose
1257.622 1263.642
1257.621 1263.641
2 × 107 3.8 × 107
K63
1.9
APG, glycation under atmospheric pressure HPG, glycation under high pressure.
Boratyński’s thermal method, up to four molecules of sugar moieties can be introduced, in contrast to ubiquitin glycated under pressure, where only a disubstituted protein was obtained. The results of our studies indicate high-pressure processing is a potential method for the preservation of foods, because the analyzed sample treated with a high pressure (
Nonenzymatic Modification of Ubiquitin under High-Pressure and -Temperature Treatment: Mass Spectrometric Studies Monika Kijewska, Karolina Radziszewska, Martyna Kielmas, Piotr Stefanowicz,* and Zbigniew Szewczuk Faculty of Chemistry, University of Wrocław, Wrocław, Poland ABSTRACT: The effect of high-pressure and/or high-temperature on the glycation of a model protein (ubiquitin) was investigated by mass spectrometry. This paper reports the impact of high pressure (up to 1200 MPa) on the modification of a ubiquitin using ESI-MS measurements. The application of glucose labeled with stable isotope allows a quantitative assessment of modification under the conditions of high-pressure (HPG) and high-temperature (HTG) glycation. A higher degree of modification was observed for the sample heated at 80 °C for 25 min under atmospheric pressure than for sample treated under high pressure. In samples treated at pressure below 400 MPa an insignificant increase of glycation level was observed, whereas high pressure (>600 MPa) has only a minor effect on the number of hexose moieties (Fru) attached to the lysine residue side chain. KEYWORDS: glycation, high-pressure glycation, Amadori products
■
INTRODUCTION High-pressure processing (HPP) preserves nutrients, natural flavor, and other sensory properties of food while extending the shelf life by preventing the growth of microorganisms by inducing structural changes to the cell membrane and inactivating important enzyme systems that disrupt metabolism of microorganisms.1,2 Some bacteria show resistance3 to pressures up to 800 MPa and or even higher. However, the application of high-pressure processing combined with increased temperature could be useful for sterilization of many products sensitive to high temperatures, such as food, cosmetics, and pharmaceuticals.4 HPP is a nonthermal preservation and pasteurization technique, which causes little or no changes in the sensory and nutritional attributes of the product being processed, unlike most conventional heat treatments.5 HPP accomplishes this by application of high hydrostatic pressures (between 100 and 1000 MPa) to food products. High pressure to about 3000 MPa does not affect covalent bonds in the molecules; therefore, many vitamins and provitamins as well as dyes, drugs; or other pharmacologically active molecules retain their unique characteristics.5,6 Bristow et al.7 and Moreno et al.8,9 reported the impact of high-pressure and alkaline conditions on the formation of Maillard products. The condensation of glyceraldehyde with diand tripeptides was hardly affected by a pressure increase (up to 500 MPa), and the reaction of glyceraldehyde, glycolaldehyde, or xylose with amino acids was slightly suppressed. For a glucose−lysine system, some stages of the Maillard reaction were affected differently.10 Staniszewska et al.11 proved that advanced glycation end products (AGEs) formed under high pressure (850 MPa) differ from those obtained thermally. In general, after high-pressure glycation (HPG), the products have a lower molecular mass than after high-temperature glycation (HTG). Immunoblotting experiments showed that the epitopes of the cross-linked glycation products formed in solution under high pressure are © 2014 American Chemical Society
not the same as those originating in dry conditions at high temperature. The antibodies against HTG products recognized HTG but not HPG products. This finding reveals that the biological properties of AGEs obtained by thermal treatment and high-pressure treatment may be different.11 The first investigations on the impact of pressure on modifications of proteinaceous amino acid residue side chains using glucose or more reactive dicarbonyl12 compounds revealed an increased level of pentosidine,13 whereas the observed formation of carboxyethylarginine from arginine did not occur at atmospheric pressure.14 In contrast, the generation of other Maillard reaction products, such as pyrraline, is hindered with the application of pressure.15 The effect of combined heat and pressure on the Maillard reaction between bovine serum albumin (BSA) and glucose was investigated by Buckow et al.16 The kinetic results showed that the protein−sugar conjugation rate increased with increasing temperature, but decreased with increasing pressure. However, five modified lysine residues were identified after 30 min of treatment at 200 MPa and 110 °C, that is, lysine residues K75, K204, K297/299, K548, and K557. Glycation of K548 was previously observed by Frolov et al.17 The effects of pressure and temperature on the reaction kinetics are antagonistic as an increase in pressure at constant temperature leads to a decrease in the entropy of the system.18 Considine et al.19 reported that BSA is relatively resistant to pressure denaturation, whereas it is easily denatured by heat and combined heat and high-pressure treatment. A similar investigation was performed on milk proteins in the absence and presence of different saccharides or dicarbonyl compounds.20 Without carbohydrates, protein cross-linking of casein is enhanced by pressure through the formation of dehydroalanine-derived lysinoalanine. In contrast, saccharide or Received: June 11, 2014 Accepted: December 18, 2014 Published: December 18, 2014 614
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 1. Deconvoluted ESI-MS spectra for ubiquitin glycated under high pressure in the range of 0.1−1200 MPa. High-Pressure Equipment. The high-pressure glycation experiments were carried out using a custom-made cylinder-piston type apparatus.28,29 The stoppered polypropylene syringe, containing the protein sample, was placed in a high-pressure vessel filled with hexane as a transmission medium and compressed (0.1−1200 MPa). High-Pressure Glycation. Ubiquitin (0.5 mg) and glucose (0.1 mg) were dissolved in 10 mmol/L aqueous ammonium formate (150 μL) at a 1:10 molar ratio and incubated at a given pressure (0.1−1200 MPa) for 24 h at room temperature (22 °C). After decompression, samples were diluted with 150 μL of a MeCN/H2O/HCOOH mixture (50:50:0.1 v/v/v). The samples were directly analyzed by ESI-MS. Additionally, the modified ubiquitin treated under a pressure of 800 MPa was hydrolyzed according to the procedure described below. High-Temperature Glycation. The high-temperature glycation of ubiquitin was performed according to Boratyński’s method.30,31 Briefly, ubiquitin and glucose or [13C6]glucose were dissolved in water at a 1:10 molar ratio and lyophilized. The dry lyophilisate was placed in an air oven at 80 °C for 25 min.27 The resulting preparation was lyophilized and directly used for MS experiments. Enzymatic Hydrolysis. Modified ubiquitin (1 mg) was dissolved in 5% aqueous formic acid (100 μL). Pepsin (10 μL of stock solution 1 mg/mL in water) was added, giving a 1:10 enzyme/substrate mass ratio. The reaction mixture was incubated for 5 h at 22 °C. The resulting digest was lyophilized and used for MS experiments. ESI-MS Measurement. ESI-MS experiments were performed using an Apex-Qe Ultra 7T instrument (Bruker Daltonic, Germany) equipped with an ESI source. The obtained fragments were subsequently analyzed by the ICR mass analyzer. The instrument was operated in the positive-ion mode and calibrated with the Tunemix mixture (Bruker Daltonic). The mass accuracy was better than 5 ppm. Spectra were recorded for 5 μmol/L protein in a MeCN/
dicarbonyl compound-induced cross-linking is reduced at high pressures. The authors hypothesized that protein cross-linking is decelerated due to either degradation of sugars or carbonyl compounds or formation of early Maillard products.20 Our recent work has been focused on the reaction of the lysine moiety with reducing sugars. The Amadori products, including glycoconjugates, are potentially useful markers of some diseases: diabetes, Alzheimer’s disease, or the aging process. We have reported an efficient method of synthesis,21 as well as selective mass spectrometric (ESI-MS) detection based on the neutral losses, borate complex formation,22,23 and isotopic (13C, 18O) labeling combined with enzymatic hydrolysis24−26 and sequencing of glycoconjugates using various methods of fragmentation (ECD, CID).27 The utilization of high pressure, an emerging technology in the area of food processing, has been increasing over the past two decades. Herein we report the impact of high pressure (up to 1200 MPa) on the level of glycation of a ubiquitin using ESIMS measurement. The quantification of the glycation level of ubiquitin was based on application of isotopic labeling [13C6].23
■
MATERIALS AND METHODS
Reagents. Reagents (including isotopically labeled [13C6]glucose; 99% 13C and pepsin) and solvents (including formic acid (HPLC grade), acetonitrile (LC-MS grade), and ammonium formate (LC-MS grade)) were purchased from Sigma. Ubiquitin was purchased from Aldrich and D-glucose from Eurochem. The reagents were used without further purification. 615
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 2. Deconvoluted ESI-MS spectrum of the mixture of ubiquitin modified thermally using [13C6]glucose and under high pressure (800 MPa) using standard glucose. H2O/HCOOH mixture (50:50:0.1 v/v/v). The sample was infused at a flow rate of 3 μL/min. The instrumental parameters were as follows: scan range, m/z 300−2500; drying gas, nitrogen; temperature of drying gas, 200 °C; potential between the spray needle and the orifice, 4.5 kV; source accumulation, 0.5 s; ion accumulation time, 0.5 s. The obtained mass spectra were analyzed using Bruker Data Analysis (Bruker Daltonic) software.
n is the number of Fru and In is the intensity of the ion. For protein samples incubated under pressure >600 MPa (Figure 1c−g), the signals corresponding to diglycated ubiquitin derivatives were observed. According to the literature, the critical pressure for denaturation of ubiquitin is approximately 600 MPa.32−34 This may explain the appearance of the doubly modified protein under higher pressure (600− 1200 MPa). The average level of modification was also calculated for 600 and 800 MPa pressures as 0.083 and 0.25, respectively. A further increase in pressure did not result in incorporation of another hexose moiety. The incubation of ubiquitin and glucose at 1000 MPa led to an additional modification, resulting in a 390.32 Da mass increase, which we were not able to identify accurately. In Figure 2, the ESI-MS spectrum of the mixture of ubiquitin modified thermally using [13C6]glucose and by a high-pressure (800 MPa) using standard glucose is presented. The signals corresponding to mono- and diglycated ubiquitin confirm the presence of protein modification resulting from the reaction with both labeled and unlabeled glucose. The same experiments were conducted to compare the level of glycation under atmospheric and high pressure (800 MPa), using regular and [13C6]glucose, respectively. In contrast to the results obtained for a high-temperature glycation using the same molar ratio of reagents as described previously,27 the application of a high pressure has minor influence on the degree of glycation. However, after the highpressure treatment, we observed additional signals in the MS spectra that were not observed after thermal treatment. We
■
RESULTS AND DISCUSSION High-Pressure Glycation. The effect of pressure on the Millard reaction between the model protein (ubiquitin) and a carbohydrate (glucose) was investigated. The series of experiments, including incubation of ubiquitin and glucose under high pressure in the range of 0.1−1200 MPa, were performed at isothermal conditions. The level of glycation was analyzed by ESI-MS. The deconvoluted mass spectra (Figure 1) reveal the signals corresponding to mono- and diglycated ubiquitin, although the spectra are dominated by the nonmodified protein. A control sample was prepared using the same molar ratio of reagents and incubated under atmospheric pressure. The peak corresponding to monoglycated ubiquitin was observed for pressures of 0.1 MPa (atmospheric) and 200 and 400 MPa (Figure 1a−c). The average level of glycation calculated according to eq 1 equals 0.08. The approximate level of glycation of a particular Lys residue was calculated by comparing intensities of ions of protein differing by the number of hexose moieties. n̅ =
∑ n*In ∑ In
(1) 616
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Figure 3. ESI-MS spectrum of the mixture of hydrolysates of ubiquitin glycated thermally (HTG) and under pressure (HPG, 800 MPa).
lower degree of modification under high pressure in comparison to the thermal modification of ubiquitin at the same protein/sugar molar ratio, the number of detected modified peptides was lower because of reduced concentration of glycated ubiquitin. We tested the levels of glycation after the pressure and thermal treatment using isotopic labeling. High-temperature glycation was performed according to Boratyński’s method using [13C6]glucose, whereas ubiquitin glycated under a high pressure was prepared using a standard glucose. The obtained modified proteins were mixed at a 1:1 weight ratio and then hydrolyzed in the presence of pepsin. The mixture of peptic fragments was analyzed directly, without a chromatographic separation, on an Apex-Ultra ESI-MS spectrometer. The same procedure was used to compare the efficiency of the glycation under atmospheric (APG) and high pressure (800 MPa) (HPG) using ubiquitin and glucose and [13C6]glucose, respectively. The Amadori products obtained by the different treatments can be identified on the basis of mass difference caused by the use of [13C6] glucose. The search for glycated peptides can be performed automatically (in-house-designed spreadsheet) or by a visual inspection, recognizing the pairs of peaks with a defined m/z difference (m/z 6 for +1 ion, 3 Th for +2 ion, etc.). In the spectrum, signals corresponding to mixtures of hydrolysates of thermally and high-pressure glycated ubiquitin are presented. In the expanded range of this spectrum (Figure 3), a pair of signals corresponding to Amadori products 5−15 and 25−45 is shown. A significant difference in the signal intensity corresponding to
were not able to identify these byproducts. Further experiments are still needed to characterize these compounds and their mechanism of formation. The regioselectivity of glycation of lysine moieties in ubiquitin was evaluated by enzymatic hydrolysis of the modified protein (incubated under pressure of 800 MPa) by pepsin followed by a direct ESI-MS measurement. Pepsin is a nonspecific protease, with the preference for peptide bonds at the C-terminus from Phe and Leu.35 The crude hydrolysate was diluted with MeCN and then analyzed by ESI-MS. The glycation under high temperature and/or high pressure is relatively nonspecific. In the sequence of ubiquitin, there are seven lysine residues and the N-terminal amino group could be also glycated. Even though there are differences in the reactivity of the particular lysine moieties, every amino group may be glycated to some extent. Therefore, the sample of a glycated protein is an extremely complex mixture. In the analyzed hydrolysate, four peptides containing Amadori products, including residues 59−67, 5−15, 4−15, and 25−45, were found. The ECD fragmentation of modified peptide confirmed their sequence and the modification sites.27 The analysis of the spectra allowed for identification of the glycated lysine residues as Lys6, Lys11, Lys27, Lys29, Lys33, and Lys63 within the sequences of ubiquitin fragments. The modifications of Lys48 and the α-amino groups, which according to the literature27 are characterized by the lowest reactivity of all the potential glycation sites in ubiquitin, were not observed. In these studies, we received four identical peptide-derived Amadori products as in the case of thermally glycated ubiquitin.23,27 Due to a much 617
DOI: 10.1021/jf505628e J. Agric. Food Chem. 2015, 63, 614−619
Article
Journal of Agricultural and Food Chemistry
Table 1. Identified Glycoconjugates in a Mixture of Hydrolysates of Ubiquitin Glycated Thermally Using Labeled Glucose ([13C6]Glucose) (HTG) and under High Pressure (800 MPa) Using Unlabeled Glucose (HPG) by MS m/z
a
glycated peptide
charge state
glycation methoda
found
calcd
intensity
modified Lys residue
4−15
1+
HPG glucose HTG [13C6]glucose
1483.864 1489.882
1483.862 1489.882
1.6 × 106 6.2 × 106
K6 or K11
3.88
5−15
1+
HPG glucose HTG [13C6]glucose
1336.794 1342.815
1336.793 1342.814
2.2 × 107 7.0 × 107
K6 or K11
3.18
25−45
2+
HPG glucose HTG [13C6]glucose
1300.210 1303.217
1300.208 1303.218
4.8 × 106 1.8 × 107
K27 or K29 or K33
3.75
59−67
1+
HPG glucose HTG [13C6]glucose
1257.623 1263.640
1257.621 1263.641
9.8 × 106 4.0 × 107
K63
4.08
intensity ratio IHTG/IHPG
HPG, glycation under high pressure; HTG, high-temperature glycation.
Table 2. Identified Glycoconjugates in a Mixture of Hydrolysates of Ubiquitin Incubated under Atmospheric Pressure Using Unlabeled Glucose (APG) and under High Pressure (800 MPa) Using Labeled Glucose ([13C6]Glucose) (HPG) by MS m/z
a
a
intensity ratio IHPG/IAPG
glycated peptide
charge state
glycation method
found
calcd
intensity
modified Lys residue
4−15
1+
APG glucose HPG [13C6]glucose
1483.863 1489.883
1483.862 1489.882
2.8 × 106 6.9 × 106
K6 or K11
2.45
5−15
1+
APG glucose HPG [13C6]glucose
1336.795 1342.814
1336.793 1342.814
3.7 × 107 7.5 × 107
K6 or K11
2.03
25−45
2+
APG glucose HPG [13C6]glucose
1300.210 1303.219
1300.208 1303.218
1.0 × 107 2.2 × 107
K27 or K29 or K33
2.2
59−67
1+
APG glucose HPG [13C6]glucose
1257.622 1263.642
1257.621 1263.641
2 × 107 3.8 × 107
K63
1.9
APG, glycation under atmospheric pressure HPG, glycation under high pressure.
Boratyński’s thermal method, up to four molecules of sugar moieties can be introduced, in contrast to ubiquitin glycated under pressure, where only a disubstituted protein was obtained. The results of our studies indicate high-pressure processing is a potential method for the preservation of foods, because the analyzed sample treated with a high pressure (
