From the Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208; and
Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom
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ABSTRACT |
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Glyoxal and glycolaldehyde are products of autoxidation of glucose or glucose adducts to proteins. Other carbohydrates, such as fructose, arabinose, and ascorbate, may also degrade to glyoxal, possibly through intermediate adducts to protein. Glyoxal may also be formed directly during oxidative degradation of polyunsaturated fatty acids (4) and during myeloperoxidase-mediated degradation of serine at sites of inflammation (5). Plasma glyoxal levels are much lower than those of glucose, but glyoxal is a far more reactive carbonyl compound. Normal plasma levels of glyoxal are reported to be 215230 nM (68) but increase to 350470 nM in diabetic subjects (7, 8), 400 nM in uraemia (6), and
760 nM in end-stage renal disease (6). Because of its high reactivity, the fraction of glyoxal bound to proteins may significantly exceed the measured glyoxal concentration in plasma.
Glyoxal is able to modify the side chains of various amino acids in protein, including those of lysine and arginine, to form several products, such as N-(carboxymethyl)lysine (CML (9)) and N
-(carboxymethyl)arginine (CMA (10)), glyoxal-derived dihydroxyimidazolidines (G-DHs; G-DH1 and G-DH2) and N
-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1), and its isomers 5-(2-amino-5-hydro-4-imidazolon-1-yl)norvaline (G-H2) and 5-(2-amino-4-hydro-5-imidazolon-1-yl)norvaline (G-H3 (11)) (Fig. 1).
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In previous work (11) on chemical modification of the model protein RNase by glucose, we concluded that CML was formed primarily by oxidation of Amadori adducts of glucose to protein and that free glyoxal, which was also formed in the reaction system, was not a significant precursor of CML. In the present study, we extend this work to analysis of the sites and products of modification of RNase by glyoxal. We show that, like the carboxymethylation of RNase, formation of G-DH and G-H in the RNase-glyoxal incubations is a site-specific process and that Arg39 and Arg85, which are the arginine residues closest to the active site of RNase, are the primary sites of modification of the enzyme. Our results indicate that dicarbonyl compounds react primarily with arginine residues in protein and that there is a high degree of specificity to the modification of both arginine and lysine residues during the Maillard reaction.
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EXPERIMENTAL PROCEDURES |
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Modification of Protein by Glucose or Glyoxal and Preparation of Tryptic Digests
RNase (13.7 mg, 1 µmol) was dissolved in 1 ml of a solution of glucose (0.4 M) or glyoxal (1 or 5 mM) in phosphate buffer (0.2 M, pH 7.4) and incubated under air at 37 °C for 3, 7, and 14 days (glucose) or 1, 3, and 7 days (glyoxal). The recovered protein was reduced with DTT, derivatized with 4-vinylpyridine, and digested with trypsin (enzyme:substrate ratio of 5:100 (w/w)) at 37 °C for 5 h. All samples were prepared in triplicate. The procedures have been published in detail elsewhere (11).
Reaction of RNase-Glyoxal Incubations with HA
Aliquots of the 7-day RNase-1 mM glyoxal incubation (40 µl, 0.04 µmol protein) were mixed with a 10-M excess of HA (carbonyl groups:amino groups = 1:5) in 0.2 M, pH 7.4 phosphate buffer (40 µl) containing diethylenetriaminepentaacetic acid (DTPA, final concentration 0.1 mM) and incubated under nitrogen for 1, 3, and 7 days at 37 °C. Control incubations used phosphate buffer in place of HA solution. Further aliquots of the 7-day RNase-1 mM glyoxal incubation (40 µl) were ultrafiltrated to remove any unreacted glyoxal (11), resuspended in an equivalent volume of 0.4 M, pH 7.4 phosphate buffer (40 µl) containing DTPA (final concentration 0.1 mM) and incubated for 1 and 7 days.
Tryptic Digestion of HA Incubations
Incubations were digested using a modification of the procedure reported by Brock et al. (11). Protein (0.5 mg) was diluted into 50 µl of 0.1 M MOPS buffer containing 6 M urea and 1 mM EDTA. DTT (0.1 µmol) dissolved in MOPS buffer:water (1:3, v/v, 5 µl) was added to the protein solution, which was flushed with nitrogen for 60 s prior to incubation at 37 °C for 3 h. 4-Vinylpyridine (2.5 µmol) mixed in methanol:water (5:2 v/v, 5 µl) was added and the protein was derivatized in the dark at room temperature for 1 h. DTT solution (35 µl, 3.5 µmol) was added to quench the reaction. The sample was diluted in water (355 µl). Sequencing grade trypsin (100 µg) was dissolved in 100 µl of 100 mM HCl, and 25 µl was added to the protein solution (enzyme:substrate ratio of 5:100 m/m), which was flushed with nitrogen and incubated at 37 °C for 5 h. Digestion was terminated by freezing at 20 °C.
Incubations of CMA with OPD and Amino Acid Analysis
CMA and OPD were each dissolved in 0.2 M, pH 7.4 phosphate buffer to give a final CMA concentration of 5 mM, and a CMA:OPD molar ratio of 1:5. DTPA was added to give a final concentration 0.1 mM. Aliquots were incubated under nitrogen at 37 °C for 1, 3, and 7 days. Amino acid analysis was conducted on a divinylbenzene cation-exchange column (3 x 250 mm) (Pickering Labs, Mountain View, CA) with a sodium citrate gradient. Amino acids were quantified by post-column fluorescence using o-phthaldehyde. CMA was quantified with reference to a standard calibration curve (010 nmol CMA).
ESI-LC-MS
Samples were fractionated on an Agilent (Palo Alto, CA) series 1100 liquid chromatograph, coupled to a Micromass (Manchester, United Kingdom) Quattro mass spectrometer (for full scan experiments) or a Micromass Q-TOF mass spectrometer (for peptide sequencing experiments). Separations were conducted using a C18 column (250 x 2 mm) and a gradient running from 0.1% aqueous acid to ACN with a flow rate of 0.2 ml/min. TFA was used as the ion-pairing reagent on the Quattro mass spectrometer and formic acid on the Q-TOF instrument. The conditions have been described in detail previously (11), and any modifications are given below.
Full-scan Experiments
Major differences in the peptide complement of samples were located by treating the entire total ion chromatogram (TIC) of each sample as a single "peak" and generating a single combined spectrum for the entire chromatogram. In a separate analysis of the data, each predicted peptide (unmodified and modified) was located within the TIC by calculating the masses of the different charged forms, extracting ion chromatograms and confirming the same retention time for each charged form, as described previously (11).
For the RNase-carbonyl incubations, estimates of the fractional modification of each peptide were determined as a percentage of the sum of all charged species of that peptide, divided by the sum of all of the charged species for the modified and unmodified peptides. This involved extracting ion chromatograms for the different charged forms of each peptide from the full-scan data and summing the peak areas for each charged form. The N-terminal or C-terminal unmodified peptide that eluted closest to the corresponding modified peptide was selected for calculations to minimize differences in mass spectrometer response between unmodified and corresponding modified peptides. For example, when Arg10 is modified, there is no cleavage by trypsin between Arg10 and Gln11. The relevant unmodified peptides are 8FER10 (8FR10) and 11QHMDSSTSAASSSNYCNQMMK31 (11QK31). Peptides 8FR10 and 11QK31 elute at 14.5 and 21.0 min, respectively, compared with a retention time of 22.5 min for the uncleaved peptide containing modified Arg10. Therefore, peptide 11QK31, which eluted closest to the modified peptide, was selected as the unmodified peptide when estimating the extent of modification at Arg10. The amount of the unmodified and each monitored modified peptide was then expressed as a percentage of the peak area for the Arg10 peptide group. The retention times of the members of each arginine peptide group that were used for calculations are shown in Table I.
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MS/MS for Peptide Sequencing
Tryptic digests were first analyzed by LC-MS with the Q-TOF operating in survey mode. CID data were collected by data-dependent switching to MS/MS mode and collection of TOF data over the mass range 1002,000 amu. Fragmentation spectra were interpreted manually.
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RESULTS |
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Location of Modified Peptides in the Glyoxal Reactions
As one approach to identifying modified peptides in the RNase-glyoxal reactions, the entire TIC, obtained using the triple quadrupole mass spectrometer for each glucose and glyoxal incubation, was treated as a single chromatographic "peak," yielding a single mass spectrum for each sample (Fig. 2, A and B). These single mass spectra were visually compared in 100 mass unit increments to locate ions that were unique to one or the other sample. Comparison of the spectra of the glucose and 5 mM glyoxal incubations at 7 days, revealed a prominent ion at m/z 982 in the glyoxal spectrum that was absent from the glucose spectrum (Fig. 2, A, B, E, and J).
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Modification at an arginine residue in a protein would lead to the formation of a tryptic peptide with a missed cleavage, because trypsin cannot cleave C-terminal to a modified arginine residue. Glyoxal is known to react with the guanidino group of both free and peptide-bound arginine to give products including hydroimidazolones (HIs (12,13)), G-DHs (14), and CMA (10, 15). The predicted mass of UK1 corresponded to the mass of the tryptic peptide with a missed cleavage C-terminal to Arg39, peptide 38DK61, where Arg39 is modified to either G-DH or CMA, which are isobaric compounds.
Confirmation of the Amino Acid Sequence of UK1 by MS/MS
To confirm the amino acid sequence of UK1, the tryptic digest of the 7-day 5 mM glyoxal incubation was analyzed by ESI-LC-MS/MS using the Q-TOF mass spectrometer. Initially, experiments were performed on the 2+ and 3+ ions of peptide 38DK61 with Arg39 modified to G-DH or CMA, but spectra were much stronger for the 3+ ion and therefore the fragmentation pattern obtained for it was analyzed in detail (Fig. 4). The data were of sufficiently high resolution for the determination of the charge state of each fragment ion and the spectrum was interpreted manually.
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We performed numerous additional MS experiments with the aim of generating additional data to confirm the identity of UK1. Both precursor ion experiments for parents of the immonium ion of G-DH or CMA at m/z 187 and multiple reaction monitoring experiments to search for the immonium ion of G-DH or CMA and the b2 ion at m/z 330, generated from peptide 38DK61 with Arg39 modified to G-DH or CMA, gave signals at the pertinent nominal masses but the identities were ambiguous due to the limited resolution of the triple quadrupole instrument. For example, the fragment at m/z 187 may also represent an a-type internal ion for DV or a b-type internal ion for AD, both of which were present in the sequence of the modified peptide. Analysis by capillary LC-MS/MS on a newer generation Q-TOF API US instrument using a limited scan range (to maximize sensitivity) and different collision energies and sample concentrations also did not reveal either ion of interest. The peptide was less amenable to analysis by MALDI-TOF/TOF than ESI-MS. The immonium ion of arginine is known to be of very low intensity (16) and it is likely that the immonium ion of the G-DH or CMA adduct of Arg39 will also be weak.
Discrimination Between G-DH, CMA, and CML
The formation of G-DH is reversible, while CMA and CML are formed irreversibly (14). To investigate whether the modified peptide in the glyoxal incubations was G-DH or CMA or both adducts on Arg39 or CML on Lys41, protein from the 7-day 1 mM glyoxal reaction was incubated with a 5-fold molar excess (based on carbonyl groups) of HA. Parallel incubations were conducted without HA, both with and without prior removal of any unreacted glyoxal. As shown in Fig. 5, the amount of UK1 decreased by 75% over a 7-day period, with a half-life of 2 days.
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The possibility that UK1 might be peptide 38DK61 with Lys41 modified to CML was ruled out because CML is extremely stable, e.g. to 6 N HCl at 95 °C for 18 h (9). The identification of UK1 as peptide 38DK61 with Lys41 modified to CML would also require that the peptide contain a missed cleavage site C-terminal to Arg39 but this unmodified peptide was not detected in tryptic digests of either the glyoxal-modified RNase incubations or of native RNase, so that cleavage of Arg39 appears to be efficient. We did detect trace levels of peptide 40CK61 with Lys41 modified to CML but UK1 was present at >100-fold higher intensity. Thus, UK1 must be peptide 38DK61 with Arg39 modified to G-DH, in line with earlier reports that dicarbonyls, such as methylglyoxal, modify arginine in preference to lysine residues on protein (13).
Fig. 5 also shows that the amount of the unmodified peptide 40CK61 increases with time of incubation with HA. Peptide 38DK61 with Arg39 modified to G-H was also located within the MS data by extracting ion chromatograms at m/z 1463, 975, and 732 for the 2+, 3+, and 4+ charged forms, respectively. This peptide also decreased on incubation with HA, but at a much slower rate than the loss of G-DH, with 80% remaining after 7 days (compared with
25% for G-DH). In control experiments, incubating the 1 mM glyoxal incubations, both with and without prior removal of unreacted glyoxal and in the absence of HA, resulted in no change in the amount of peptide 38DK61 in which Arg39 was modified to G-DH. Thus, the discharge of G-DH adducts was dependent on reaction with HA.
Site Specificity and Kinetics of Formation of G-DH and G-H Adducts
We have demonstrated that although CML forms at every lysine when RNase is incubated with glucose, the distribution of CML is not uniform and Lys41 is the major site of modification (11). Therefore, we anticipated that G-DH and G-H might also form at other arginine residues in RNase, i.e. Arg10, Arg33, and Arg85, in addition to Arg39, and that their distribution among the arginine residues might be uneven. We therefore searched for ions with calculated m/z values of the different charge states of the predicted peptides, 8FERQHMDSSTSAASSSNYCNQMMK31 (8FK31), 32SRNLTK37 (32SK37) and 67NGETNCYQSYSTMSITDCRETGSSKYPNCAYK98 (67NK98), where Arg10, Arg33, and Arg85 were modified, respectively, to G-DH or G-H. In previous studies, we observed that trypsin produced almost no cleavage between Lys91 and Tyr92 of RNase. Therefore, modifications of peptide 67NK98, with two missed cleavage sites, were monitored. Peptides 8FK31 and 67NK98, containing, respectively, Arg10 and Arg85 modified to G-DH and G-H, were located by extracting ion chromatograms from the full-scan data, thus establishing the formation of both of these adducts at Arg10 and Arg85. When the 7-day 1 mM glyoxal reaction was incubated with HA, the kinetics of loss of peptides containing Arg10 and Arg85 modified to G-DH/CMA were similar to those of peptide 38DK61 with Arg39 modified to G-DH, indicating that G-DH and not CMA was also the major or sole adduct on these arginine residues. Peptide 32SK37 with Arg33 modified to either G-DH or G-H was not detected in the RNase-glyoxal reactions, despite a strong signal for the native peptide 34NLTK37, indicating that Arg33 was not converted to these adducts in detectable amounts in the glyoxal incubations, even after 7 days in 5 mM glyoxal.
Although G-H exists as three structural isomers, we could detect only one peak within the LC-MS data corresponding to G-H on Arg10, Arg39, and Arg85, possibly because the isomers were not resolved on the LC column or because some of them were present below the limit of detection. The 3+ ions of the G-DH adducts of peptides 8FK31 and 67NK98, and the G-H adduct of peptide 38DK61 were also detected in the single combined spectrum that was extracted from the full-scan data of the glyoxal incubations, but not from the glucose incubations (Fig. 2, D, F, I, and K), and their retention times match those expected for these adducts. None of these modified peptides was present in sufficiently high concentrations for peptide sequencing experiments.
The kinetics of formation of the peptides containing arginine residues, together with the kinetics of loss of the relevant unmodified peptides in the 1 mM glyoxal incubations, are shown in Fig. 6. Similar graphs were obtained for the 5 mM glyoxal incubations, but with a 2- to 3-fold increased response. Arg39 and Arg85 were identified as the major sites of adduct formation.
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DISCUSSION |
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Estimating Amounts of Modifications
Quantitation of absolute and relative modification of proteins by LC-MS/MS is a major challenge. We have previously estimated the extent of modification of different lysine residues in RNase by calculating relative amount (RA) values (11), i.e. the area units for charged species of the modified peptide, compared with the area units of the C-terminal peptide of RNase. One advantage of this technique is that values are normalized between samples, but a disadvantage is that the mass spectrometer response to different tryptic peptides of native RNase is variable, limiting the validity of the technique for comparing amounts of different tryptic peptides. The use of RA values to estimate modifications is more meaningful when the number of amino acid residues in the unmodified and modified peptides is the same or similar; in this case the mass spectrometer response is likely to be similar. Biemel and Lederer (17) established that lysine, fructoselysine, and N6-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)-L-lysinate gave comparable responses when analyzed by ESI-LC-MS. They also provided evidence that these modifications on an identical peptide resulted in an almost equivalent mass spectrometer response. Therefore, this approach was applied in this study to monitor the loss of the G-DH and G-H adducts at Arg39 within peptide 38DK61 and regeneration of peptide 40CK61 in the HA reactions. These peptides differed in length by only two amino acid residues and also possessed very similar retention times on the HPLC column (Table I).
To follow the kinetics of formation of G-DH and G-H adducts at the different arginine residues within RNase, we expressed the data as a percentage of the unmodified and modified peptides within each arginine peptide group. This method eliminated variations in RA values due to inter-sample and inter-batch variations in the derivatization/digestion procedure and LC-MS response.
Identification of Peptide 38DK61 Containing Arg39 Modified to G-DH
Based on reversibility of modification by treatment with HA, UK1 was identified as primarily (>75%) peptide 38DK61 containing the G-DH adduct at Arg39. Glomb and Lang (14) monitored the degradation of authentic G-DH in the presence of OPD or aminoguanidine (AG) at 37 °C and pH 7. These experiments yielded half-lives of 12 days for G-DH, consistent with the half-life we obtained following incubation of our 7-day 1 mM glyoxal incubations with HA. CMA has been identified in collagen incubated in 1 M glucose (10), and it has been demonstrated, using ESI-LC-MS, that levels of CMA on serum proteins from diabetic subjects are significantly higher than those from age-matched controls (15). G-DH was not measured in either of these studies but, based on our data and those of Glomb and Lang (14), it may be present at higher concentrations than CMA.
Site Specificity of G-DH and G-H Formation
As shown previously (11), the formation of CML in RNase-glucose systems is site-specific (11), occurring primarily at Lys41 in the active site and derived primarily from the Amadori adduct. Based on the present work, modification of protein by glyoxal is also site-specific, occurring primarily on Arg39 and Arg85 in RNase, with small amounts of modifications forming on Arg10, but no detectable adducts on Arg33. In previous work, Arg39 and Arg85 were also identified as the main sites of modification of RNase by -dicarbonyls (18). The earliest work involved modification of bovine RNase A by phenylglyoxal, which also yielded a reversible adduct (19). Later studies, involving the incubation of RNase with 1,2-cyclohexanedione (20, 21) and 3-ethoxy-2-ketobutanal (22), concluded that Arg39 and Arg85 were the most modified sites, followed by Arg10, with Arg33 being unreactive. The adduct formed from 1,2-cyclohexanedione, i.e. N7,N8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine, was characterized (20) and, like the glyoxal adduct, G-DH, its formation was reversible on incubation with HA. The lack of reactivity at Arg33 was attributed to hydrogen-bonding to Asp14 (19, 22).
Relative Amounts of AGEs on Proteins
The data presented here show that, in the glyoxal incubations, the order of abundance of the adducts was G-DH > G-H > CML. Only low levels of CML peptides were formed from glyoxal and, in the glucose incubations, no G-DH or G-H adducts could be detected. We have previously reported (11) that, in the glucose incubations, the main route to CML is by oxidation of the ARP. This is confirmed by the absence of G-DH or G-H adducts on the glycated protein, indicating that little glyoxal was formed, and the higher abundance of peptide 40CK61 carboxylated at Lys41 in the glucose incubation compared with the glyoxal system.
AGEs are potential biomarkers of various disease states, including diabetes, atherosclerosis, and neurodegenerative disease. Relative amounts of different AGEs in physiological proteins and fluids may differ according to the severity of disease or particular tissue, reflecting local differences in carbonyl formation and oxidative stress. In general, HI adducts are reported to be present at higher concentrations than CML on plasma and tissue proteins (23, 24). For example, G-H1 has been reported to be present at about twice the level of CML on plasma proteins of healthy human subjects. Levels of G-H1 on retina, nerve, and plasma proteins of streptozotocin-induced diabetic rats also increased significantly compared with those of normal controls (24), reflecting elevated levels of plasma glyoxal in diabetes. Little attention has been given to G-DH adducts on protein (14), but our studies suggest that they may be present at higher concentrations than either CMA or G-H.
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CONCLUSIONS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, September 17, 2004, DOI 10.1074/mcp.M400002-MCP200
1 The abbreviations used are: AGE, advanced glycation end-product; AG, aminoguanidine; CMA, N-(carboxymethyl)arginine; CML N
-(carboxymethyl)lysine; DTPA, diethylenetriaminepentaacetic acid; G-DH, glyoxal-derived dihydroxyimidazolidine; G-DH1, N
-(4,5-dihydroxy-4,5-dihydro-1-imidazolidinyl)ornithine; G-DH2, 5-(4,5-dihydroxy-2-imino-1-imidazolidinyl)norvaline; G-H glyoxal-derived hydroimidazolone; G-H1, N
-(5-hydro-4-imidazolon-2-yl)ornithine; G-H2, 5-(2-amino-5-hydro-4-imidazolon-1-yl)norvaline; G-H3, 5-(2-amino-4-hydro-5-imidazolon-1-yl)norvaline; HA, hydroxylamine; HI, hydroimidazolone; OPD, o-phenylenediamine; RA, relative amount; TIC, total ion chromatogram; UK1, Unknown 1.
* This study was funded by the Wellcome Trust, via a Short-Term Travel Grant 06564/Z/01/Z (to J. M. A.) and by National Institutes of Health Grant DK-19971 (to J. W. B.).
¶ To whom correspondence should be addressed: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading, RG6 6AP, United Kingdom. Tel.: 44-118-931-8730; Fax: 44-118-931-0080; E-mail: j.m.ames{at}reading.ac.uk
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REFERENCES |
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