Arginine-Derived Advanced Glycation End Products Generated in

Apr 11, 2014 - AGEs are considered to be toxic in vivo due to their ... Here, we studied the formation of arginine-related AGEs in peptides under cond...
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Arginine-Derived Advanced Glycation End Products Generated in Peptide−Glucose Mixtures During Boiling Andrej Frolov,† Rico Schmidt,† Sandro Spiller, Uta Greifenhagen, and Ralf Hoffmann* Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy and Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany S Supporting Information *

ABSTRACT: Glycation refers to the reaction of amino groups, for example in proteins, with reducing sugars. Intermediately formed Amadori products can be degraded by oxidation (Maillard reactions) leading to a heterogeneous class of advanced glycation end-products (AGEs), especially during exposure to heat. AGEs are considered to be toxic in vivo due to their pronounced local and systemic inflammatory effects. At high temperatures, these reactions have been mostly investigated at the amino acid level. Here, we studied the formation of arginine-related AGEs in peptides under conditions simulating household cooking at physiological D-glucose concentrations. High quantities of AGE-modified peptides were produced within 15 min, especially glyoxal-derived products. The intermediately formed dihydroxy-imidazolidine yielded glyoxal- (Glarg) and methylglyoxal-derived hydro-imidazolinones (MG-H), with Glarg being further degraded to carboxymethyl-L-arginine (CMA). Carboxyethyl-L-arginine was not detected. The formation rates and yields were strongly increased in the presence of physiologically relevant concentrations of Fe(II)-ions and ascorbate. A nearby histidine residue increased the content of AGEs, whereas glutamic acid significantly reduced the CMA levels. KEYWORDS: AGEs, food processing, Glarg, glycation, methylglyoxal

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Nδ-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-orni-thine(tetrahydroargpyrimidine).13 Arginine-derived AGEs are produced endogenously and can be ingested during feeding (exogenous source) especially after heating.14 They have been suggested as markers of diabetes mellitus,15 arteriosclerosis,16 uremia,17 and aging.18 Mammals absorb exogenous AGEs relatively fast, and these derivatives can be detected in blood already a few hours after consumption.19 Their interaction with receptors of advanced glycation end-products (e.g., RAGE) results in enhanced nuclear factor κB (NF-κB)-mediated expression of inflammation response genes and the development of oxidative stress in tissues.20 As this phenomenon contributes to arteriosclerotic changes in organs,21 AGE formation and degradation pathways during food processing is an urgent healthcare research area. At the amino acid level, Glomb’s group studied the pathways of in vitro formation and degradation of Arg-derived AGEs at 37 °C for Nα-tert-butoxycarbonyl (Boc)-arginine with glyoxal8 or methylglyoxal.22 Such processes have not been studied at higher temperatures mimicking cooking of proteins, which can be simulated by peptides representing denatured proteins, to the best of our knowledge. Here, we incubated model peptides containing a single arginine residue at 95 °C with physiologically relevant concentrations of D-glucose (25 mmol/L) in phosphate buffer. The obtained glycation products were identified by electrospray ionization (ESI) mass spectrometry (MS) and the kinetics of

INTRODUCTION Glycation (or nonenzymatic glycosylation) refers to the reversible reaction of amino groups in proteins, peptides, and lipids with reducing sugars.1 This nonenzymatic post-translational modification affects the N-termini of proteins and the εamino groups of lysine residues. Aldoses and ketoses can react with primary amino groups to yield Amadori2 and Heyns3 products, respectively. The sugar moieties in these early glycation products, as well as the free aldoses and ketoses, can be degraded by oxidation to reactive dicarbonyl intermediates, such as glyoxal and methylglyoxal.1 Both compounds can modify both lysine and arginine side chains yielding many products that are typically called advanced glycation end-products (AGEs).4 As arginine residues are often localized in active, allosteric, and metal-binding centers of enzymes,5 their glycation may directly affect the cell status and thus the physiology of the corresponding organs and tissues.6 Several glyoxal- and methylglyoxal-derived AGEs have been reported for arginine residues (Figure 1). Incubation of βcasein with glyoxal yields 1-(4-amino-4-carboxybutyl)2-imino5-oxo-imidazolidine (Glarg),7 which slowly hydrolyzes under physiological conditions to acid-labile carboxymethylarginine (CMA).8 Methylglyoxal produces dominantly Nδ-(5-methyl-4oxo-5-hydroimidazolinone-2-yl)-L-ornithine (MG-H1)9 and at lower quantities 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon-1-yl)pentanoic acid (MG-H2) and 2-amino-5-(2amino-4-hydro-4-methyl-5-imidazolon-1-yl)pentanoic acid (MG-H3).10 At pH 8.0 and room temperature (RT), MG-H3 is quantitatively hydrolyzed to carboxyethyl-L-arginine (CEA) within 3 days.11 Additionally, the imidazolinones can react with a second methylglyoxal molecule yielding Nδ-(5-hydroxy-4,6dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine)12 and © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3626

November 28, 2013 March 30, 2014 April 2, 2014 April 11, 2014 dx.doi.org/10.1021/jf4050183 | J. Agric. Food Chem. 2014, 62, 3626−3635

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Figure 1. Structures and mass increments of glyoxal- (A, B) and methylglyoxal-derived (C−G) arginine-AGEs: Glarg (A), CMA (B), MG-H1 (C), MG-H2 (D), MG-H3 (E), CEA (F), and argpyrimidine (G). to 4 h) under continuous shaking (450 rpm) in the absence or presence of D-glucose (25 mmol/L). Incubations were stopped by addition of aqueous EDTA (6 mmol/L, 10 μL) on ice and stored at −80 °C. RP-HPLC-ESI-QqTOF-MS and MS/MS. Samples corresponding to an initial amount of 90 pmol peptide were separated on a Aqua C18column using a HPLC 1100 system (Agilent Technologies, Böblingen, Germany) consisting of a binary gradient pump (G1312A), online micro degasser (G1379A) and Marathon autosampler (10 μL full loop injection, Spark Holland B.V., Emmen, Netherlands). Eluents A and B were water and acetonitrile, respectively, containing formic acid (0.1%, v/v). Elution was achieved by isocratic conditions (3% eluent B, 5 min) followed by two consecutive linear gradients to 40% (3 min) and 95% eluent B (23 min) using a flow rate of 220 μL/min. LC-MS/MS relied on a shallower gradient (from 4% to 30% eluent B in 4 min and then to 37% eluent B in 14 min). The ESI-QqTOF-MS was equipped with a TurboIon Spray source (QSTAR Pulsar I; AB Sciex, Darmstadt, Germany) and operated in positive ion mode using the Analyst QS 1.0 software (AB Sciex, Darmstadt, Germany). Ion spray voltage and declustering potential were set to 5000 and 50 V, respectively. Curtain, nebulizing, and drying gases were set to 30 psig. The ion source was held at 450 °C. Spectra were acquired with automatically adjusted pulsing frequency and 1 s accumulation time. Collision-induced dissociation (CID) MS/MS experiments relied on low Q1 resolution, collision potentials of 40−70 V, and nitrogen as collision gas (5 psig). In MS mode the instrument was calibrated with a mixture of glycine, sucrose, and maltoheptaose (250 μmol/L each, 60%, v/v aq. acetonitrile) using the [MH + Gly]+ ions of sucrose (m/z 418.1555) and maltoheptaose (m/z 1228.4196). Calibration of the tandem mass spectra relied on y1- (m/z 175.195) and y11-ions (m/z 1285.545) of the doubly protonated [Glu-1] fibrinogen peptide 1. Relative quantification was performed by integration of extracted ion chromatograms (XICs, ± 0.1 m/z-units) at specific retention times.

their formation and degradation was studied for a period of 4 h. Additionally, the effects of reactive oxygen species (ROS) and nearby amino acid residues were evaluated.

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MATERIALS AND METHODS

Chemicals. Suppliers and their products were as follows: Iris Biotech GmbH (Marktredwitz, Germany): 9-fluorenylmethoxycarbonyl (Fmoc-) rink amide AM resin and Fmoc-L-Arg(Pbf)−OH (peptide synthesis grade); ORPEGEN Pharma (Heidelberg, Germany): all other Fmoc-protected L-amino acids (peptide synthesis grade); Biosolve (Valkenswaard, Netherlands): N,N′-dimethylformamide (DMF, ≥99.8%), piperidine (≥99.5%), and dichloromethane (DCM, ≥99.9%); Carl Roth GmbH & Co KG (Karlsruhe, Germany): trifluoroacetic acid (TFA, 99.9%), α-D-glucose hydrate (≥99.5%), potassium hydrogen phosphate (≥99%), and potassium dihydrogen phosphate (≥99%); VWR International GmbH (Dresden, Germany): diethyl ether (100%) and acetonitrile (≥99.9%). All other chemicals were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Water was purified in-house (resistance 18 mΩ/cm) on a PureLab Ultra Analytic System (ELGA Lab Water, Celle, Germany). Peptide Synthesis. Peptides Ac-ASGAGSARASGXASA-NH2 with X = Ala (1), X = His (2), and X = Glu (3) were synthesized on a multiple peptide synthesizer Syro2000 (MultiSynTech GmbH, Witten, Germany) by Fmoc/tert-butyl-chemistry using eight equivalents (equiv) of Fmoc-amino acid derivatives (or acetic acid for N-terminal acetylation) activated with DIC/HOBt in DMF.23 Peptides were cleaved with TFA at RT (2 h), precipitated with diethyl ether, and purified by RP-HPLC using formic acid or TFA (0.1% v/v) in water (eluent A) and 60% (v/v) aqueous acetonitrile (eluent B) using a gradient slope of 1% eluent B/min. The peptides were aliquoted (32 nmol) in 1.5-mL safe lock tubes (Eppendorf AG, Hamburg, Germany), dried under vacuum, and stored at −20 °C. In vitro Glycation Reactions with Model Peptides. Peptide aliquots were reconstituted in potassium phosphate buffer (0.1 mol/L, pH 7.4, 0.1 mL) containing iron(II) sulfate (0, 18, or 590 μmol/L) and ascorbic acid (0, 60, or 1200 μmol/L), and incubated (95 °C, up 3627

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RESULTS Identification of Arginine-Derived Glycation Products. When peptide 1 was incubated with D-glucose and Fe(II)SO4 at 95 °C, a total of eight peptide products with lower or higher masses than the original peptide were detected by RPHPLC-ESI-QqTOF-MS already after 15 min of exposure time (Table 1). Reaction products were numbered in their elution Table 1. Retention Times, Precise Masses, and ArginineDerived AGE-Modified Peptides Identified during the Incubation of Peptide 1 with D-Glucose (25 mmol/L) in the Presence of Fe(II)SO4 (18 μmol/L) no.

tR (min)

1 1-1 1-2 1-3 1-4 1-5 1-6 1-7 1-8

18.6 18.3 18.5 18.5 18.5 18.8 18.8 19.0 19.0

a

m/z [M + 2H] 616.831 595.802 636.805 645.816 656.801 645.817 656.803 643.821 652.818

± ± ± ± ± ± ± ± ±

2+

0.006 0.002 0.003 0.002 0.003 0.001 0.002 0.017 0.002

Δm

b

+0.00 −42.06 +39.95 +57.97 +79.94 +57.98 +79.94 +53.98 +71.97

residue in position 8

mass accuracy (ppm)

arginine ornithine Glarg GD-HI argpyrimidine CMA argpyrimidine MG-H MGD-HI

46.2 17.4 7.9 16.7 22.3 18.2 19.2 20.9 7.5

a

Average m/z-value and standard deviation recorded during different experiments. bModification-specific mass shift relative to the increment mass of an arginine residue.

Figure 2. Total ion current chromatogram (A), extracted ion chromatogram (m/z 636.8 ± 0.1; small insert), and tandem mass spectrum of the precursor ion at m/z 636.8 (B) representing Glargcontaining peptide ASGAGSARGlargASGAASA.

order and identified by their exact m/z values and tandem mass spectra (Table 1 and Figure 2). The tandem mass spectra typically displayed intense signals of the b-ion series indicating that the mass shift was located at Arg8, while in some cases it was deduced from the exact mass shift of a peptide fragment (Supporting Information (SI) Figure S-1). The increment masses revealed three glyoxal- (Glarg, GD-HI, and CMA) and two methylglyoxal-derived modifications (MG-H and MGDHI) as well as ornithine (loss of urea). Furthermore, two signals with m/z-values corresponding to an argpyrimidine-modified peptide were detected at different retention times (Table 1). Neither cross-linked products (dimers) nor oxidized arginine residues, such as hydroxy-arginine or glutamate-5-semialdehyde, were observed.24 Some modified peptides displayed characteristic fragment ions that further supported the deduced structures. Glarg- (1-2) and MG-H-containing peptides (1-7, Figures 2B and SI S-1B) as well as the corresponding hydrated forms (1-3 and 1-8, respectively) displayed signals at m/z 152.1 and 166.1. Most likely, these signals were related to the arginine immonium ion (Im, m/z 129.1) and represented the corresponding Im-NH3 fragment (m/z 112.1) containing Glarg and MG-H moieties (mass shifts of 40 and 54 m/z-units, respectively). If this assumption is correct, then the signals would represent hydroimidazolinone and dihydroxyimidazolidine and thus confirm 1-3 and 1-8 as glyoxal- and methylglyoxal-derived dihydroxy-imidazolidines (GD-HI and MGD-HI), respectively (Table 1, Figure 1). As the MG-H-modified peptide eluted in one peak in RPC using different eluent compositions (SI Figure S-2), it remains open, if it was a single MG-H-modified peptide or a mixture of different MG-H-versions. Based on the absence of the signal at m/z 152.1 in the tandem mass spectrum of peptide 1-5, this analyte was annotated as CMA (Table 1).

CEA was neither detected here nor in any of the other incubation mixtures. Kinetics of Arginine-Related AGEs. Unmodified peptide 1 was very stable when incubated with iron(II) sulfate (18 μmol/L, 95 °C). Basically no degradation products were detected by RPC-MS. The peak area, which was calculated from the XICs, decreased only to ∼86% after 4 h (t test: p = 0.14; Figure 3A). The degradation rate was significantly higher in the presence of D-glucose, with 46% of the initial peptide amount left after 1 h and only 5% after 4 h. Obviously, the peptide loss was related to glucose oxidation25 yielding glyoxal- and methylglyoxal-derived AGEs or oxidative degradation of the peptide backbone.26 As described above, the incubation yielded eight modified peptides in reasonable quantities (Table 1), which could be separated by RPC and MS and thus be quantified from their XICs. It should be noted that Arg-derived modifications with different gas phase basicity could influence the ionization properties of AGE-modified peptides significantly. Thus, only signal intensities of structurally related peptides can be used to estimate their relative quantities at different time points. The detected amount of the GD-HI-modified peptide (1-3) increased for 45 min and decreased afterward almost to background level after 4 h (Figure 3B). The Glarg-peptide (12) quantity increased during the first hour and was basically stable within the error range afterward (Figure 3B). The same was true for the CMA-adduct (1-5). Considering the slow degradation of the unmodified peptide, it can be concluded that Glarg- or CMA-modified peptides were either not further produced after 1 h or they were immediately degraded upon formation. 3628

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Figure 3. Degradation of peptide 1 (A, ⧫) when incubated with (solid) and without (dotted) D-glucose (25 mmol/L) Fe(II)SO4 (18 μmol/L) and formation of the detected AGE products (B−D). Aliquots were analyzed after 0, 15, 30, 60, and 240 min by RPC-ESI-QqTOF-MS in positive ion mode. Presented are the peak areas calculated by integrating the XICs of the doubly protonated quasimolecular ions (±0.10 m/z-units) containing a glyoxal-derived residue (B), i.e. Glarg (▲), GD-H1(●), and CMA (■), a methylglyoxal-derived residue (C), i.e. MG-H (▲) and MGD-H1 (●), or another reaction product (D), i.e. ornithine (■) and argpyrimidine (1-4 ▲, 1-6 ●).

decreased to 60% after 4 h incubation (SI Figure S-3). In the presence of glucose, all three peptides demonstrated very similar degradation kinetics and their amounts decreased to about 5% after 4 h. Interestingly, the kinetics of all three glyoxal-derived products, i.e. Glarg (i-2, i corresponds to the peptide number), GD-HI (i-3), and CMA (i-5) depended on the substitution in position 12 (Figure 4A−C). All three peptides showed very similar formation kinetics of Glarg and GD-HI during the first 30 min. The content of the Glarg-modified peptide 2 (Glu) decreased afterward and was near background levels at the end of the incubation period. The amounts of the corresponding Ala- and His-containing peptides increased further up to 60 min before they decreased slowly with the Glarg-modified peptide 3 being always present at high levels (Figure 4A). The GD-HImodified products showed a similar profile, but here peptides 1 and 2 showed similar kinetics reaching the maximal amounts after 45 min, whereas the quantities of the GD-HI-modified peptide 3 increased further for the next 15 min and then decreased relatively fast afterward. Surprisingly, CMA-modified peptides showed a different behavior with peptide 3 forming very small amounts of the CMA-product, whereas peptide 2 was modified at higher levels than peptide 1 between 1 and 4 h (Figure 4C). Unexpectedly, the substitutions had only minor effects on the kinetics of both methylglyoxal products (Figure 4D and E), although His might favor the formation of MG-H at the beginning. The only difference was seen for the MGD-HIcontaining peptides in the last 3 h of the incubation period, when peptide 2 was degraded faster than peptide 3. Thus, our

The two methylglyoxal-derived modifications, i.e. MG-H (17) and MGD-HI (1-8), showed similar kinetics as GD-HI with increasing amounts up to around 1 h and a decrease afterward (Figure 3B and C). The degradation rates from 1 to 4 h were slightly higher than that for the unmodified peptide (Figure 3), but lower than that for the GD-HI peptide. The signal intensities of the methylglyoxal-derived products were 10-fold lower than for the corresponding glyoxal-derived peptides. The formation and degradation of the ornithine-containing peptide (1-1) resembled the kinetics for MG-H and to a lower extent that of Glarg (Figure 3D). Interestingly, the two products annotated as argpyrimidine (1-4 and 1-6) showed different kinetics with the earlier eluting compound being produced fast within the first 15 min, and then slightly increased up to 1 h before it slowly decreased. Peptide 1-6 increased continuously for 1 h and decreased 20% during the following 3 h period, which resembled the kinetics of GD-HI (Figure 3). Thus, the two products annotated as argpyrimidine must have different structures and should be formed and degraded by different reactions. Further studies are required to identify their stereochemical structures and origins. Influence of Nearby Residues on Formation of ArgDerived AGEs. Up to this point, only one inert peptide sequence was studied containing a single reactive arginine residue in position eight. Thus, we extended our study to two highly conserved peptides by substituting Ala-12 with His (peptide 2) or Glu (peptide 3). These peptides were incubated with glucose and iron sulfate in conditions identical to those described for the alanine-containing analogue. Peptide 3 was as stable as peptide 1 (∼80%), whereas the content of peptide 2 3629

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Figure 4. Formation and degradation curves of Glarg- (i-2, A; i denotes the peptide number), GD-HI- (i-3, B), CMA- (i-6, C), MG-H- (i-8, D), and MGD-HI-modified (i-9, E) peptides 1 (□), 2 (◊), and 3 (○). Peptides were incubated with D-glucose (25 mmol/L) and Fe(II)SO4 (18 μmol/L) at 95 °C. Samples were analyzed by RPC-ESI-QqTOF-MS in positive ion mode after incubation periods of 0, 15, 30, 60, and 240 min. Intensities were calculated as peak areas by integrating the XICs (n = 3) of the doubly protonated quasimolecular ions (±0.10 m/z-units).

findings give support for stabilizing effect of His and a destabilizing effect of Glu on MGD-HI (Figure 4E). Effect of ROS on the Formation and Degradation of Arginine-Derived AGEs. After analyzing the formation and degradation of AGEs under weak oxidation conditions (18 μmol/L Fe(II)SO4), the influence of lower and higher iron(II)concentrations as well as the presence of ascorbate at two different concentrations was tested for peptide 1. Based on the above results the incubation period was reduced to 195 min, as the signals of all AGEs reached the highest intensities after about 1 h. Additionally, the incubation mixtures were analyzed at six time points within the first hour to describe the quantitative changes more precisely. In the absence of Dglucose, the unmodified peptide was degraded by approximately 50% within the first hour with only 5% left after 195 min of incubation. In the presence of D-glucose, the peptide was always degraded at very similar rates mostly independent of the Fe(II) and ascorbate concentrations. Higher Fe(II) concentrations appeared to favor the degradation slightly, whereas ascorbate accelerated it at low Fe(II) concentrations (3.3-fold molar access of ascorbate) and reduced it for high Fe(II) concentrations (2-fold molar access of ascorbate) (Figure 5A).

The Glarg-modified peptide (1-2) was obtained at around 20% lower quantities in the absence of iron(II) and approximately 70% higher quantities at high iron(II) concentrations relative to the original conditions. Addition of ascorbate significantly increased Glarg formation at low iron(II) concentration conditions, but decreased its formation at high iron(II) concentration (Figure 5B). The maximal amount of Glarg was detected in all samples after approximately 1 h. The GD-HI-modified peptide (1-3) showed basically a similar dependence on the iron(II) and ascorbate concentrations as the Glarg-containing peptide (Figure 5B and C) with the maximal product quantities obtained after 45 to 60 min. Afterward the peptide amounts decreased significantly and reached nearly identical levels at the end of the incubation period. Formation of the CMA-modified peptide (1-5) depended on the iron(II) and ascorbate concentrations as described above for Glarg and GD-HI. The highest levels were again obtained after 1 h. The degradation, however, depended on the presence of ascorbate. Without ascorbate the product amounts were stable from 60 to 195 min, whereas they decreased significantly in the presence of ascorbate (Figure 5D). 3630

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Figure 5. Time-course curves of unmodified (1, A), Glarg- (1-2, B), GD-HI- (1-3, C) and CMA-modified peptides (1-5, D) observed after incubation of peptide 1 with D-glucose and different concentrations of iron(II) and ascorbate. Peptide 1 was incubated at 95 °C with D-glucose (25 mmol/L) in phosphate buffer (▲) containing 18 μmol/L Fe(II)SO4 (■), 590 μmol/L Fe(II)SO4 (●), 18 μmol/L Fe(II)SO4 and 60 μmol/L ascorbate (□), or 590 μmol/L Fe(II)SO4 and 1.2 mmol/L ascorbate (○). Samples were analyzed by RPC-ESI-QqTOF-MS in a positive ion mode after incubation periods of 0, 5, 10, 15, 30, 60, and 195 min. Intensities were calculated as peak areas by integrating the XICs (n = 3) of the doubly protonated quasimolecular ions (±0.10 m/z-units).

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DISCUSSION

rich food. The iron(II) concentration was similar to the levels found in blood and tissue.30 Qualitative Analysis of Arg-Derived AGEs. The pattern of identified glyoxal-derived products (GD-HI, Glarg, and CMA) matched well to data reported previously for amino acids.8 As we observed only one symmetrical chromatographic peak for MG-H (SI Figure S-4), it remains unclear if only one of the three reported MG-H-isomers was produced or several that were not resolved by RP-HPLC. At the amino acid level, this separation is achieved significantly easier than in a 15residue-long peptide, where the relatively polar MG-H will only slightly contribute to the retention. Thus, further studies using shorter peptides are necessary to provide a better chromatographic separation. Recently MGD-HI was proposed as an intermediate of MG formation using methylglyoxal and Boc-protected arginine.22 This compound was indeed detected here and tentatively identified by its characteristic MS/MS fragmentation pattern. Most likely it is more stable within a peptide or protein sequence than at the amino acid level, which would indicate an influence of the α-carboxyl-group on the product stability. In spite of ornithine not being considered as an AGE product, it is a well-known marker of Arg-related AGEs in proteins.31 Here, we could show that Arg can lose urea at significant levels and that the obtained Orn is relatively quickly degraded afterward, most likely through glycation of its δamino group. Nevertheless, this possibility was not further investigated.

Peptide Sequence and Incubation Conditions. To simplify the data analysis and to obtain mostly Arg-derived modifications at a single position, the peptide sequence consisted only of Ala, Gly, and Ser. These three amino acids are relatively inert against oxidation and do not react with intermediately formed aldehydes, such as glyoxal and methylglyoxal. At the same time they are relatively polar and thus guarantee a good solubility, even when the guanidinogroup is transformed to a hydrophobic AGE. The only reactive groups would be the N-terminus, which could even be glycated, and the C-terminus. To avoid any degradation at the termini, these were blocked by acetylation and amidation, respectively. Additionally, Arg was flanked by two inert Ala-residues, which contain only a side-chain methyl-group that is unlikely to interfere sterically with reactions at the guanidino-group. Finally and very important for deducing AGE-modified peptides, the tandem mass spectra of model peptide Ac-ASGAGSARASGAASA-NH2 (1) displayed an almost complete b-series at similar intensities all over the mass spectrum (SI Figure S-1A). Therefore, although proline would prevent ordered structures, it was excluded from the sequence, to avoid any influence on the fragmentation pattern and interference with modificationspecific signals.27 The selected D-glucose concentration corresponded to the highest levels occurring in mammals,28,29 which represent a good landmark for the maximal “natural” sugar load in protein3631

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Figure 6. Proposed pathways of the formation of arginine-related peptide glyoxal- (A) and methylglyoxal- (B) derived glycation end-products.

The fragmentation patterns of AGE-containing peptides by CID displayed many internal fragment ions, which were not observed for the unmodified peptide and thus can be considered as characteristic for AGE-modified peptides containing hydroimidazolinone or dihydroxyimidazolidine

groups (SI Figure S-1B−E and Table S-1). These signals likely originated from the nucleophilic attack of the imino nitrogen on a carbonyl C atom of the peptide backbone32 These internal fragments and the immonium ions (Im-NH3) distinguished isobaric dihydroxyimidazolidine from carboxymethylated or 3632

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but their signal intensities and thus their quantities were around 5−10 times lower than those for Glarg and GD-HI, respectively. This difference can be explained by the higher glyoxal concentrations in comparison to methylglyoxal during glucose degradation.33 Therefore, a lesser amount of MG-H was produced than Glarg during the first 15 min (Figure 2C). The degradation of Glarg and MG-H occurred at similar rates indicating that they are equally stable and their spectral decrease results mostly from peptide degradation. The same appears to be true for MGD-HI and GD-HI (Figure 3B and C). The mechanisms of hydroimidazolinone formation and degradation were comprehensively evaluated by incubating Boc-Arg-OH with methylglyoxal.22 The authors proposed the formation of MG-H3 through a dihydroxy-imidazolidine precursor, but could not detect it. This intermediate was, however, detected in tryptic digests of human plasma treated with methylglyoxal in vitro,34 indicating, in line with our findings, that MGD-HI is more stable in peptides and proteins than in Boc-Arg-OH. At neutral pH MG-H3, its hydrolysis product CEA, and MG-H1 form an equilibrium.22 As we detected only MG-H, the CEA-product was present at significantly lower levels (below the detection limit) indicating that the equilibrium was shifted to the most stable product MG-H1. This would also explain why we detected only a single MG-H peak, corresponding to MG-H1 whereas MG-H3 was below the detection limit. Alternatively, MG-H3 could be further processed, for example to argpyrimidine. In this case analogue 1-7 would also correspond to MG-H1. This could be clarified by NMR, but NMR spectroscopy requires considerably larger amounts of samples. This was not further investigated, as the functional effects of MG-H do not appear to depend upon the isomer. The increasing amounts of the Orn-containing peptide confirm the proposed equilibrium between AGE formation and cleavage,31 which may involve a ring-opening reaction with a subsequent nucleophilic attack of the guanidine nitrogen on the β-C atoms of CMA or CEA. Among the two products annotated as argpyrimidine, only 1-6 accumulated over time as reported previously for Boc-Arg-OH at 37 °C.22 The intensities of argpyrimidine and MG-H pseudomolecular ions were quite similar, though glucose is slowly degraded to methylglyoxal under the applied conditions.33 Assuming similar ionization efficiencies for both peptides, our findings indicate that argpyrimidine but not CEA was the main product of MG-H transformation. Thus, the detected hydroimidazolone was presumably MG-H3, which reacted more or less quantitatively with a second methylglyoxal molecule to yield argpyrimidine (peptide 1-6). Based on the kinetics of 1-6 and 1-7 it can be concluded that either (i) MG-H is not the only precursor of argpyrimidine, or (ii) at least one relatively stable MG-Hderived intermediate of argpyrimidine formation exists though not detected here (Figure 6B). Neighbor Group Effect. In vitro and in vivo data indicate that basic and acidic residues in the i+4 position (nearest side chain in an α-helix) modulate both Schiff base formation and Amadori-rearrangement35,36 whereas such neighbor group effects have not been investigated for Arg-derived AGEmodifications. Thus, we studied the influence of Glu- and His-substitutions at position 12 (i+4 relative to Arg-8). As the basic or acidic residue altered the ionization efficiencies relative to the original peptide, the peak areas were normalized to the preincubation peak area of each peptide (SI Figure S-3).

carboxyethylated species. The patterns of these signals in the product ion mass spectra of GD-HI-modified peptide 1-3 (m/z 645.8) and the Glarg-modified peptide 1-2, for example, were quite similar (SI Figures S-1C and S-2B, respectively). In contrast, the tandem mass spectrum of CMA-modified peptide 1-5 did not display any of these internal fragments (SI Table S1). Thus, the increment mass of 214 m/z-units indicated CMA at position 8 (SI Figure S-1E). The two weak signals at m/z 656.8 eluting at 18.5 and 18.8 min were annotated as argpyrimidine-containing peptides (1-4 and 1-6, Table 1), but the tandem mass spectra did not provide sufficiently intense signals to deduce their structures. Obviously, the low levels of methylglyoxal produced by oxidation of glucose reduced the probability that the guanidino group can react with two dicarbonyl molecules before the intermediate undergoes further modifications. This confirms earlier reports that the reaction of MG-H3 with a second methylglyoxal molecule requires high dicarbonyl concentrations and long incubation times at the amino acid level.22 Our data collected under physiological conditions indicate, however, that these AGE-modifications will be produced at very low levels in vivo. Mechanistic Considerations of AGE-Formation and Degradation. Although signal intensities in ESI-MS depend on many factors and are difficult to judge even for closely related sequences, several Arg-derived AGE modifications have closely related structures and properties, and thus show similar ionization properties in peptides. This was shown, for example, for the model peptide and its Glarg- and MG-H2-modified versions that were displayed in the mass spectra at very similar intensities (SI Figure S-5). Only the carboxyl groups of CMA and CEA should significantly weaken their ionization. For hydroimidazolone-related AGE-modifications, the relative signal intensities are expected to be closely dependent upon their concentrations (SI Figure S-4). Thus, it is possible to quantify not only the signal intensity of a given AGEmodification at different time points, but also the relative contents of chemically related products, i.e. Glarg, GD-HI, MGH, MGD-HI, and argpyrimidine. The Glarg-modified peptide 1-2 showed a degradation time curve identical to that of its dihydroxyimidazolidine precursor GD-HI (1-3), though at a higher intensity after 30 min (Figure 3B). The spectral intensities of the CMA-modified peptide 1-5 were just below those of 1-2, but considering the suppressive effect of the negatively charged carboxyl-group of CMA, peptide 1-5 was probably present at significantly higher quantities than 1-2 (Glarg) and especially 1-3 (GD-HI). The delayed appearance of 1-5 (CMA) relative to 1-2 (Glarg) and the earlier degradation of 1-3 (GD-HI) might indicate that GDHI is the precursor of Glarg, which is further processed to CMA. This assumption is in full agreement with a mechanism proposed by Glomb and Lang at the amino acid level.8 The authors observed a 50% conversion of Glarg to CMA within 6 h at 37 °C. The higher temperatures applied here should accelerate the reaction around 60-fold considering that chemical reactions are typically accelerated 2-fold when increasing the reaction temperatures by 10 °C. Thus, hydrolytic cleavage of the hydroimidazolone ring of Glarg appears to be the dominant pathway of CMA formation, though the ringopening reaction of GD-HI might represent a minor pathway (Figures 3B and 6A). Methylglyoxal-derived products MG-H (peptide 1-7) and MGD-HI (peptide 1-8) showed the same time dependence as the above-discussed glyoxal-derived AGEs (Figure 3B and C), 3633

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should be noted, however, that the content of these AGEmodified peptides decreased for longer heating periods, although it is unclear if only the peptide was further degraded leading to a heterogeneous mixture of small peptides and amino acids not detectable by RPC-ESI-MS or if the AGE modification itself was eliminated along with cleavage of the peptide chain. Since this issue remains to be answered to evaluate the potential toxicity of AGEs produced during cooking, further research is needed to characterize the formation and degradation of peptide- and protein-related AGEs using conditions more closely mimicking household food cooking protocols.

His substitution yielded higher Glarg quantities, while the negatively charged Glu reduced them (Figure 4A). Both observations can be explained by the proton-catalyzed formation of Glarg.8 The protonated His residue should favor this reaction whereas the deprotonated Glu residue would disfavor it. This His effect was also obtained for GD-HImodified peptide 3 (Figure 4B). Considering additionally the kinetics of the Glarg-containing peptides (Figure 4A), Glu most likely suppressed the dehydration process of GD-HI accompanied by Glarg formation (Figure 6A). Remarkably, the basic and acidic residues did not affect the rate of GD-HI formation. Hence, the stabilizing effect that prevents degradation of Glargand CMA-containing peptides might be more important than the catalytic effect. Interestingly, this stabilization was also observed for MGD-HI, but not for its direct product MG-H (Figure 4E and D, respectively). The presence of Glu drastically reduced the quantities of CMA (Figure 4C), most likely by forming an ion pair with Glarg. This would increase the electron density of the hydroimidazolone ring in position C-5 and thus reduce the strength of the nucleophilic addition of a hydroxide, the reaction of which initiates the bond cleavage between C4 and C5 to convert Glarg into CMA. Additionally, the negative charge of the carboxylate might also prevent the nucleophilic attack of the hydroxide. Variation of Oxidation Conditions. As the initially chosen incubation conditions yielded dominantly glyoxalderived AGEs (peptides 1-2, 1-3, and 1-5), the oxidation conditions were varied assuming that ROS production levels might influence peptide degradation rates and AGE patterns. Thus, the H2O2-driven Fenton reaction37 was studied for trace, low, and high iron(II) concentrations, and enhanced by ascorbate-mediated Fe(III) reduction.38,39 The higher peptide degradation rates observed for low additive concentrations (Figure 5A) may indicate a dual role of ascorbate with pro-oxidative effects at low concentrations and antioxidative properties at high concentrations.38 Expectedly, the opposite effects were obtained for the AGE modifications (Figure 5B−D). Most probably, iron(II) had a pro-oxidative effect by accelerating metal-catalyzed autoxidation of glucose and thus favored the production of α-dicarbonyls (glyoxal and methylglyoxal),25 whereas ascorbate had only a low influence. Remarkably, GD-HI (1-3) was produced much faster than Glarg (1-2) and CMA (1-3), which both showed similar formation rates. This correlates to the reportedly faster glucose oxidation caused by higher ROS quantities25 and thus higher levels of glyoxal. Remarkably, both Glarg- and CMA-containing peptides were stable in the absence of iron(II) and only slowly degraded in the presence of iron(II). The addition of ascorbate increased the degradation of both peptides strongly, very similar to that of GD-HI that was very labile under all conditions. Our observations clearly indicate that glycoxidation is a relatively fast process with many products already formed after short periods of elevated temperatures. Whereas the peptides were relatively stable in the absence of sugars when heated under air atmosphere, the addition of D-glucose drastically accelerated peptide degradation including the formation of seven different AGE products, a process that was even more pronounced in the presence of low iron(II) concentrations, a condition characteristic to commercial meat products. Hence, cooking of protein-rich food in the presence of sugars can produce presumably toxic compounds within a few minutes. It

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ASSOCIATED CONTENT

S Supporting Information *

Table S-1, Modification-specific internal fragment ions of peptide 1; Figure S-1, Tandem mass spectra of the unmodified peptide 1 and its AGE-modified products; Figure S-2, Initial peak areas of peptides 1, 2, and 3 calculated from XICs; Figure S-3, Stability of peptides 1 to 3 at 95 °C; Figure S-4, Reversedphase and extracted ion chromatograms of peptide 1; Figure S5, Signal intensities of peptide H-AGSARASGFA-NH2 and related AGEs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 (0) 341 9731330. Fax: +49 (0) 341 9731339. E-mail: hoff[email protected]. Author Contributions †

Authors A.F. and R.S. contributed equally in the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the “Deutsche Forschungsgemeinschaft” (DFG, HO-2222/7-1), the European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony), the “Bundesministerium für Bildung and Forschung” (BMBF), and a stipend to U.G. provided by the Ernst Schering Foundation is gratefully acknowledged.

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REFERENCES

(1) Ulrich, P.; Cerami, A. Protein glycation, diabetes, and aging. Recent Prog. Horm. Res. 2001, 56, 1−21. (2) Hodge, J. E. The Amadori rearrangement. Adv. Carbohydr. Chem. 1955, 10, 169−205. (3) Heyns, K.; Noack, H. Die Umsetzung von D-Fructose mit LLysine und L-Arginin und deren Beziehung zu nichtenzymatischen Bräunungsreaktionen. Chem. Ber. 1962, 95, 720−727. (4) Grillo, M. A.; Colombatto, S. Advanced glycation end-products (AGEs): involvement in aging and in neurodegenerative diseases. Amino Acids 2008, 35, 29−36. (5) Betts, M. J.; Russell, R. B. Amino acid properties and consequences of substitutions. In Bioinformatics for Geneticists; Barnes, M. R., Gray, I. C., Eds.; John Wiley & Sons, Ltd: New York, 2003; pp 289−316. (6) Ahmed, N.; Thornalley, P. J.; Dawczynski, J.; Franke, S.; Strobel, J.; Stein, G.; Haik, G. M. Methylglyoxal-derived hydroimidazolone advanced glycation end-products of human lens proteins. Invest. Ophthalmol. Vis. Sci. 2003, 44 (12), 5287−5292. (7) Schwarzenbolz, U.; Henle, T.; Haeßner, R.; Klostermeyer, H. On the reaction of glyoxal with proteins. Z. Lebensm. Unters. Forsch. A 1997, 205, 121−124. 3634

dx.doi.org/10.1021/jf4050183 | J. Agric. Food Chem. 2014, 62, 3626−3635

Journal of Agricultural and Food Chemistry

Article

(8) Glomb, M. A.; Lang, G. Isolation and characterization of glyoxalarginine modifications. J. Agric. Food Chem. 2001, 49 (3), 1493−1501. (9) Henle, T.; Walter, A. W.; Haeßner, R.; Klostermeyer, H. Detection and identification of a protein-bound imidazolone resulting from the reaction of arginine residues and methylglyoxal. Z. Lebensm. Unters. Forsch. 1994, 199, 55−58. (10) Ahmed, N.; Argirov, O. K.; Minhas, H. S.; Cordeiro, C. A.; Thornalley, P. J. Assay of advanced glycation endproducts (AGEs): Surveying AGEs by chromatographic assay with derivatization by 6aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nε-carboxymethyl-lysine- and Nε-(1-carboxyethyl)lysine-modified albumin. Biochem. J. 2002, 364 (Pt 1), 1−14. (11) Gruber, P.; Hofmann, T. Chemoselective synthesis of peptides containing major advanced glycation end-products of lysine and arginine. J. Pept. Res. 2005, 66 (3), 111−124. (12) Shipanova, I. N.; Glomb, M. A.; Nagaraj, R. H. Protein modification by methylglyoxal: Chemical nature and synthetic mechanism of a major fluorescent adduct. Arch. Biochem. Biophys. 1997, 344 (1), 29−36. (13) Oya, T.; Hattori, N.; Mizuno, Y.; Miyata, S.; Maeda, S.; Osawa, T.; Uchida, K. Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts. J. Biol. Chem. 1999, 274 (26), 18492−18502. (14) Henle, T. Protein-bound advanced glycation end-products (AGEs) as bioactive amino acid derivatives in foods. Amino Acids 2005, 29 (4), 313−322. (15) Ahmed, N.; Thornalley, P. J. Advanced glycation endproducts: What is their relevance to diabetic complications? Diabetes, Obes. Metab. 2007, 9 (3), 233−245. (16) Price, C. L.; Knight, S. C. Advanced glycation: A novel outlook on atherosclerosis. Curr. Pharm. Des. 2007, 13 (36), 3681−3687. (17) Thornalley, P. J.; Rabbani, N. Highlights and hotspots of protein glycation in end-stage renal disease. Semin. Dial. 2009, 22 (4), 400− 404. (18) Ahmed, N.; Thornalley, P. J.; Dawczynski, J.; Franke, S.; Strobel, J.; Stein, G.; Haik, G. M. Methylglyoxal-derived hydroimidazolone advanced glycation end-products of human lens proteins. Invest. Ophthalmol. Vis. Sci. 2003, 44 (12), 5287−5292. (19) Koschinsky, T.; He, C. J.; Mitsuhashi, T.; Bucala, R.; Liu, C.; Buenting, C.; Heitmann, K.; Vlassara, H. Orally absorbed reactive glycation products (glycotoxins): An environmental risk factor in diabetic nephropathy. Proc. Natl. Acad. Sci. U. S. A 1997, 94 (12), 6474−6479. (20) Goh, S. Y.; Cooper, M. E. Clinical Review: The role of advanced glycation end products in progression and complications of diabetes. J. Clin. Endocrinol. Metab. 2008, 93, 1143−1152. (21) Schmidt, A. M.; Stern, D. M. RAGE: A new target for the prevention and treatment of the vascular and inflammatory complications of diabetes. Trends Endocrinol. Metab. 2000, 11 (9), 368−375. (22) Klopfer, A.; Spanneberg, R.; Glomb, M. A. Formation of arginine modifications in a model system of Nα-tert-butoxycarbonyl (Boc)-arginine with methylglyoxal. J. Agric. Food Chem. 2011, 59 (1), 394−401. (23) Rich, D. H.; Singh, J. The Carbodiimide Method. In The Peptides; Gross, E., Meinenhofer, J., Eds.; Academic Press: New York, 1979; pp 241−261. (24) Amici, A.; Levine, R. L.; Tsai, L.; Stadtman, E. R. Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions. J. Biol. Chem. 1989, 264 (6), 3341−3346. (25) Wolff, S. P.; Dean, R. T. Glucose autoxidation and protein modification. The potential role of “autoxidative glycosylation” in diabetes. Biochem. J. 1987, 245 (1), 243−250. (26) Berlett, B. S.; Stadtman, E. R. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 1997, 272 (33), 20313−20316. (27) Papayannopoulos, I. A. The interpretation of collision-induced dissociation tandem mass spectra of peptides. Mass Spectrom. Rev. 1995, 14, 49−73.

(28) Fox, A.; Eastwood, C.; Gentry, C.; Manning, D.; Urban, L. Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat. Pain 1999, 81 (3), 307−316. (29) Shimomura, I.; Bashmakov, Y.; Ikemoto, S.; Horton, J. D.; Brown, M. S.; Goldstein, J. L. Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. U. S. A 1999, 96 (24), 13656−13661. (30) Jacobs, A.; Miller, F.; Worwood, M.; Beamish, M. R.; Wardrop, C. A. Ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. Br. Med. J. 1972, 4 (5834), 206−208. (31) Sell, D. R.; Monnier, V. M. Conversion of arginine into ornithine by advanced glycation in senescent human collagen and lens crystallins. J. Biol. Chem. 2004, 279 (52), 54173−54184. (32) Bythell, B. J.; Csonka, I. P.; Suhai, S.; Barofsky, D. F.; Paizs, B. Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine. J. Phys. Chem. B 2010, 114 (46), 15092−15105. (33) Phillips, S. A.; Thornalley, P. J. The formation of methylglyoxal from triose phosphates. Investigation using a specific assay for methylglyoxal. Eur. J. Biochem. 1993, 212 (1), 101−105. (34) Kimzey, M. J. Identification, characterization, and quantification of dicarbonyl adducts in the plasma proteome in type-2 diabetes. Doctoral Dissertation. The University of Arizona, Department of Pharmaceutical Sciences: Tucson, AZ, 2011. (35) Venkatraman, J.; Aggarwal, K.; Balaram, P. Helical peptide models for protein glycation: Proximity effects in catalysis of the Amadori rearrangement. Chem. Biol. 2001, 8 (7), 611−625. (36) Zhang, Q.; Monroe, M. E.; Schepmoes, A. A.; Clauss, T. R.; Gritsenko, M. A.; Meng, D.; Petyuk, V. A.; Smith, R. D.; Metz, T. O. Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects. J. Proteome Res. 2011, 10 (7), 3076−3088. (37) Jomova, K.; Vondrakova, D.; Lawson, M.; Valko, M. Metals, oxidative stress and neurodegenerative disorders. Mol. Cell. Biochem. 2010, 345 (1−2), 91−104. (38) Buettner, G. R.; Jurkiewicz, B. A. Catalytic metals, ascorbate and free radicals: combinations to avoid. Radiat. Res. 1996, 145 (5), 532− 541. (39) Bollineni, R. C.; Fedorova, M.; Hoffmann, R. Identification of carbonylated peptides by tandem mass spectrometry using a precursor ion-like scan in negative ion mode. J. Proteomics 2011, 74 (11), 2351− 2359.

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