Bioconjugate Chem. 2003, 14, 619−628
619
Site-Specific Quantitative Evaluation of the Protein Glycation Product N6-(2,3-Dihydroxy-5,6-dioxohexyl)-L-lysinate by LC-(ESI)MS Peptide Mapping: Evidence for Its Key Role in AGE Formation Klaus M. Biemel* and Markus O. Lederer Institut fu¨r Lebensmittelchemie (170), Universita¨t Hohenheim, Garbenstrasse 28, D-70593 Stuttgart, Germany. Received December 13, 2002
Advanced glycation end products (AGEs) contribute to various pathologies associated with the general aging process and long-term complications of diabetes. Involvement of R-dicarbonyl intermediates in the formation of such compounds is firmly established. We now report on the first unequivocal identification of the dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysinate (4) on lysozyme via its quinoxaline derivative N6-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)-L-lysinate (6), formed by reaction of 4 with o-phenylenediamine (OPD). For accurate quantification of the total content of 6 as well as of glucosepane 5 by LC-(ESI)MS, 13C6-labeled reference compounds were independently synthesized; 5 so far is the only established follow-up product of 4. With an overall lysine derivatization quota of 5%, compound 4 is shown to be a quantitatively important Maillard intermediate of which only about 8‰ are transformed into the cross-link 5. Hence, the major follow-up products of the highly reactive intermediate 4 are yet unknown. The site-specific quantitative evaluation of aminoketose 1 and quinoxaline 6 by LC-(ESI)MS peptide mapping shows that all lysine moieties in lysozyme are in fact modified by these compounds. If an arginine side chain is adjacent to the lysine moiety, transformation of 1 into 4 seems to be favored. The efficient formation and high reactivity of 4 clearly points to its potential as exogenous or endogenous glycotoxin.
INTRODUCTION
The spontaneous reaction of reducing carbohydrates with amino functions, preferentially the -amino group of lysine moieties in peptides and proteins, is generally subsumed under the terms Maillard reaction or nonenzymatic browning (1, 2). The reaction is initiated by the formation of Schiff bases which, as a rule, rearrange to the more stable aminoketoses (Amadori products); in the course of this process the protein becomes ‘glycated’. The first steps of the glycation reaction are reversible. After about 20-40 days, a steady state is reached for the Amadori product 1 (Figure 1), i.e., its concentration remains constant and is only dependent on the glucose concentration (3). During prolonged reaction, the aminoketoses are degraded to a plethora of compounds, designated ‘advanced glycation end products’ (AGEs).1 R-Dicarbonyl compounds such as 3-deoxyglucosone (3DG, 2; Figure 1), methylglyoxal (MG), and glyoxal (GO) are established key-intermediates in these complex reac* To whom correspondence should be addressed. Phone: +49 711 459 4095. Fax: +49 711 459 4096. E-mail: bipa@ uni-hohenheim.de. 1 Abbreviations used: AG, aminoguanidine; AGE, advanced glycation end product; t-Boc, tert-butoxycarbonyl; 3-DOG, 3-deoxyglucosone; DTT, dithiothreitol; glucosepane 5, 6-[2-{[(4S)-4ammonio-5-oxido-5-oxopentyl]amino}-6,7-dihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]azepin-4(5H)-yl]-L-norleucinate; GO, glyoxal; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; ISTD, internal standard; LC-(ESI)MS, coupled liquid chromatography - electrospray ionization mass spectrometry; MG, methylglyoxal; MWCO, molecular weight cutoff; OPD, o-phenylenediamine; SIM, selected ion monitoring; TFA, trifluoroacetic acid; TIC, total ion current; TLCK, NR-ptosyl-L-lysine chloromethyl ketone; TPCK, N-p-tosyl-L-phenylalanine chloromethyl ketone.
Figure 1. Structural formulas of the Amadori compound 1, the R-dicarbonyl intermediates 2-4, and the major protein cross-link glucosepane 5. The dideoxyosone N6-(2,3-dihydroxy5,6-dioxohexyl)-L-lysinate (4a,b) generally is incorporated into a protein and thus differs strikingly from 3-deoxyglucosone (2); compound 4 is the established precursor of glucosepane 5. So far, formation of N6-(5,6-dihydroxy-2,3-dioxohexyl)lysinate (3) from glucose could not be proven.
tion cascades which proceed both in vitro and in vivo. Such compounds may be regarded as so-called ‘glycotoxins’, capable of modifying proteins and lipoproteins by AGE formation. The pathogenicity of endogenous, glucosederived AGEs in human tissues has been subject of intense investigation and is now well established (4-7).
10.1021/bc025653e CCC: $25.00 © 2003 American Chemical Society Published on Web 04/01/2003
620 Bioconjugate Chem., Vol. 14, No. 3, 2003
As reported in the recent literature, the pluripotent effects of AGEs range from multiple gene activation to proatherosclerotic and glomerulosclerotic effects involving cytokine and growth factor modulation, lipid peroxidation, and albuminuria (6-10). Of particular significance is the finding that the process of degradation of tissue-bound AGEs exposes a new pool of previously internal, highly reactive AGE intermediates to circulation. Some of these serum AGE precursors have been found capable to react with proteins (e.g., LDL, collagen), propagating oxidative modifications or forming new AGE cross-links (11, 12). The formation of covalently crosslinked proteins is a major consequence of the advanced Maillard reaction. Especially in long-lived tissue proteins, the cross-links are supposed to be the most significant AGEs, as they are likely responsible, for example, for the decreased flexibility of collagen (13) and the high level of urea-insoluble proteins in human cataracts (14). Koschinsky et al. found that AGE precursors may not only be derived from endogenous processes but also from dietary uptake (15). This study confirmed the absorption of 10% of ingested AGEs and showed that only one-third of those resorbed are excreted within 48 h in the urine of patients with normal renal function. Exogenous glycotoxins, not cleared by the kidney, are supposed to remain biologically active and thus modify tissue proteins. Especially in case of nephropathy, exogenous as well as endogenous AGE precursors are of pathophysiological significance. Since most of the structurally identified AGEs (e.g., CML (16), GOLD (17), CEL (18), MOLD (19), imidazolone A (20), pyrraline (21)) are derived from R-dicarbonyl intermediates, it seems reasonable to expect that glycotoxins liberated from glycated proteins incorporate a similar highly reactive building block. The only proteinlinked dicarbonyl structure which so far has been considered in terms of AGE formation is N6-(5,6-dihydroxy2,3-dioxohexyl)lysinate (3, Amadori dione) (6, 22) even though it has not yet been identified in proteins, either in vitro or in vivo. Recently, we could show that the major protein cross-link glucosepane 5 is derived from the regioisomeric structure N6-(2,3-Dihydroxy-5,6-dioxohexyl)L-lysinate (4) (23, 24). In this dideoxyosone, being formed via the aminoketose 1, the lysine N is directly bonded to C-1 of the original sugar; generation of 4 requires carbonyl shifts along the entire carbohydrate backbone. We now report on the site-specific quantitative evaluation of the aminoketose 1 and the lysine-linked dicarbonyl compound 4 in lysozyme by LC-(ESI)MS peptide mapping using several enzymes for proteolytic digestion. For quantification of the total content and unequivocal identification of 4 by LC-(ESI)MS, a 13C-labeled standard for its quinoxaline derivative 6 was synthesized independently and the structure definitely established. The effect of the concentration of the trapping reagent o-phenylenediamine (OPD) on glucosepane 5 suppression and quinoxaline 6 formation was also investigated. EXPERIMENTAL PROCEDURES
Materials. Milli-Q water (purified to 18 MΩ‚cm-2; Millipore, Eschborn, Germany) was used in the preparation of all solutions, and HPLC grade methanol and acetonitrile were employed for LC-MS. Propionic acid, 2-propanol, o-phenylenediamine (OPD), NR-t-Boc-L-lysine, N-t-Boc-L-lysine, NR-t-Boc-L-arginine, dithiothreitol (DTT), and iodoacetamide were purchased from Fluka (NeuUlm, Germany), n-heptafluorobutyric acid (HFBA) from
Biemel and Lederer Table 1. 13C NMR Data for the Labeled Positions of the Quinoxaline 6a,b-13C6 (D2O, 25°C)
δ (ppm)a 6a C-1 C-2 C-3 C-4 C-5 C-6
50.3 69.3 72.2 39.6 155.1 146.9
J (Hz)b 6b 49.9 70.2 73.0 39.6 155.1 146.9
1J 1,2 1J 2,3 1J 3,4 1J 4,5 1J 5,6 2J 4,6
6a
6b
39.5 40.5 38.0 50.5 35.5 7.0
39.0 41.0 37.0 50.5 35.5 7.0
a δ (ppm), chemical shift for the indicated carbon. The NMR data of the unlabeled compounds 6a,b are given in ref 24. The numbering C-1 to C-6 refers to the original carbohydrate backbone and does not follow IUPAC rules. b J (Hz), coupling constant between the indicated carbons.
Aldrich (Steinheim, Germany), phosphate-buffered saline (PBS), R-chymotrypsin (TLCK treated), endoproteinase Glu-C (V 8 strain), trypsin (TPCK treated), peptidase, and aminopeptidase M from Sigma (Steinheim, Germany), D-glucose, trifluoroacetic acid (TFA), and Pronase E from Merck (Darmstadt, Germany), and D-glucose-13C6 from Dr. Glaser AG (Basel, Switzerland). For a phosphate buffer salt mixture giving solutions with pH 7.4, KH2PO4 (2.68 g, 20 mmol) and Na2HPO4‚2H2O (14.30 g, 80 mmol) were mixed vigorously. N2-(tert-Butoxycarbonyl)N6-(1-deoxy-β-D-fructopyranos-1-yl)-L-lysine (t-Boc-1) was generously supplied by Marcus A. Glomb (Technical University of Berlin, Germany). Glucosepane-13C6, was prepared according to ref 23. Preparation of N6-(2,3-Dihydroxy-4-quinoxalin-2ylbutyl)-L-lysinate (6a,b)-13C6. Synthesis with D-glucose-13C6 and isolation of the quinoxaline 6a,b-13C6 follows the procedure given in ref 24; for NMR data see Table 1. Incubations of Nr-t-Boc-L-lysine and Glucose with OPD. D-Glucose (90 mg, 0.5 mmol), was reacted with NRt-Boc-L-lysine (8.6 mg, 35 µmol), OPD (2.7 mg, 25 µmol, 10.8 mg, 100 µmol, or 27 mg, 250 µmol), and DTPA (2 mg, 5 µmol) at pH 7.4 in phosphate buffer (5 mL, 0.1 M). The mixtures were flushed with argon and kept for 5 days at 60 °C. Aliquots (100 µL) were taken, hydrochloric acid (6 N, 150 µL) was added, and the solution was kept at ambient temperature for 15 min. The volume was filled up to 0.5 mL, the pH adjusted to 7 by slowly adding solid NaHCO3, and the mixture subjected to LC-(ESI)MS analysis (column A, full scan mode). D-Glucose-Hen Egg Lysozyme Incubations. Lysozyme (1 g, 70 µmol) and D-glucose (1.8 g, 10 mmol) were dissolved in phosphate buffer (100 mL, 0.1 M, pH 7.4) containing DTPA (78 mg, 0.2 mmol) and passed through a 0.45 µm membrane filter. To four aliquots (15 mL each) was added OPD (0 mg, 0 mmol; 8.1 mg, 75 µmol; 32.4 mg, 300 µmol; and 81.0 mg, 750 µmol, respectively). The samples were filtered (sterile filter, 0.2 µm) into tubes, sealed tightly, and kept for 8 weeks at 37 °C with gentle shaking. Each lysozyme incubation (equivalent to 150 mg of protein) was transferred to an Amicon 8050 (Witten, Germany) stirred cell, diluted to 30 mL, ultrafiltered
Localization of AGE Precursor on Protein
(Millipore PM 10 filter disks, MWCO 10 kDa) with water (0.6 L) by applying a pressure of 4.5 bar, and lyophilized. Total Enzymatic Hydrolysis of Lysozyme. Aliquots of proteins (1 mg) were enzymatically hydrolyzed according to the procedure of Glomb et al., protocol C (25). A stock solution (20 µL) containing 6a,b-13C6 (10.7 mg/L) and 5a-d-13C6 (4.5 mg/L) was added at the outset of the digestion procedure. The hydrolysates were ultrafiltered by centrifugation (Millipore Ultrafree MC, MWCO 5 kDa) and the filtrates (0.4 mL) subjected to LC-(ESI)MS analysis (column A, SIM mode). Evaluation of the Relative Molar ESI+ Responses of Lysine, 1, and 6. To a solution (300 µL) containing lysine, 1, and 6 (14 mM each) was added hydrochloric acid (3 N, 2.7 mL) and the mixture kept at room temperature for 15 min. The pH was adjusted to 7 with solid NaHCO3, and the solution was diluted 1:50 with water and immediately subjected to LC-(ESI)MS analysis (column A, SIM mode). Lysozyme Carbamidomethylation. Native or incubated lysozyme (15 mg, 1.05 µmol) was dissolved in Tris-HCl buffer (3 mL, 250 mM, pH 8.5) containing 8 M urea and 1 mM EDTA. A fresh solution of DTT (170 µL, 100 mM) was added, the tubes were flushed with argon, and the reduction was allowed to proceed in the dark at 37 °C for 30 min. Iodoacetamide solution (75 µL, 1 M) was added and the mixture kept at 37 °C for 30 min, followed by addition of precooled acetone (10 mL, -20 °C), vigorous shaking, and centrifugation (2500g, 0 °C). The resulting pellets were washed thrice with cold acetone/water (80:20 v/v), suspended in water (3 mL), and lyophilized. Trypsin, Chymotrypsin, and Endoproteinase Glu-C Peptide Mapping. Three separate proteolytic digestions were performed for the carbamidomethyl derivatives of native lysozyme and lysozyme incubated in the presence of 100 mM D-glucose and 20 mM or 50 mM OPD. For the tryptic and chymotryptic digestion, the protein (2 mg) was dissolved in ammonium carbonate buffer (250 µL, 100 mM, pH 8.5), aliquots (5 µL) of TPCKtreated trypsin or TLCK-treated chymotrypsin (both 5 mg/mL, in 1 mM HCl containing 12 mM CaCl2) were added, and the mixture was kept at 37 °C for 2 h. For the endoproteinase Glu-C digestion, the protein (2 mg) was dissolved in 200 µL phosphate buffer (50 mM, pH 8.7), endoproteinase Glu-C solution (16 µL, 4 mg/mL) added, and the mixture kept at 37 °C for 18 h. The digestions were stopped by adjusting the pH to 5 with 10% formic acid. After incubation (30 min, 37 °C) with gentle shaking, the hydrolysates were adjusted to pH 3 and lyophilized. The residues were dissolved in the eluent for peptide mapping (80 µL) and subjected to LC-(ESI)MS analysis (column B, full scan mode). LC-(ESI)MS Analyses. The LC-(ESI)MS analyses were run on an HP1100 (Hewlett-Packard, Waldbronn, Germany) HPLC system coupled to a Micromass (Manchester, UK) VG platform II quadrupole mass spectrometer equipped with an electrospray (ESI) interface. The HPLC system comprised an HP1100 autosampler, HP1100 gradient pump, HP1100 thermoregulator, and HP1100 diode array detector (DAD) module. Columns: (A) YMC - Pack Pro C18, 120 Å, 5 µm, (guard column 10 × 4.6 mm, column 150 × 4.6 mm); column temperature 25 °C; flow rate 1.0 mL/min; (B) Waters (Eschborn, Germany) XTerra MS C18, 5 µm (guard column 20 × 3.0 mm, column 250 × 3.0 mm); column temperature 25 °C; flow rate 0.5 mL/min. The following gradient % MeOH (t [min]) was applied for column A: 10 mM HFBA-MeOH, 5(0)-50(25)-95(30-35)-5(40-47). MS parameters: ESI+,
Bioconjugate Chem., Vol. 14, No. 3, 2003 621 Scheme 1. Transformation of Dideoxyosone 4 into the Quinoxaline 6a
a Intermediate 4 cannot directly be detected in complex incubation mixtures and thus is reacted with the efficient trapping reagent OPD to yield the stable derivative 6.
source temperature 120 °C, capillary 3.5 kV, HV lens 0.5 kV; the system was operated either in full scan (m/z 1501000, cone 40 V) or in SIM mode (span 0.5 Da; dwell time 0.2 s). The monitoring masses in the SIM mode were m/z 435.3, 429.3 (cone 65 V) and m/z 369.25, 363.25 (cone 45 V) for the quantification of 6, or m/z 147.0, 309.1, and 363.2 (cone 40 V) for the evaluation of the relative molar response of lysine, 1, and 6. The separation of the lysozyme peptides from the digests was performed on column B with 0.1% aqueous TFA-MeCN/H2O/TFA (90: 10:0.1, v/v/v) and the following gradient: % (MeCN/H2O/ TFA (90:10:0.1, v/v/v)) (t [min]), 2(0)-44(35)-100(3739)-2(42-48). The MS system was operated in the full scan mode (m/z 240-1500), cone voltage ramp 20-80 V. For the quantitative evaluation of modified peptides and acquisitions in the selected ion monitoring (SIM) mode, postcolumn addition of propionic acid/2-propanol (3:1, v/v) in a 1:3 ratio with the HPLC mobile phase was performed with a Knauer WellChrom Maxi-Star K-1000 HPLC pump. The eluent was finally split 1:20 before being introduced into the ion source. Nuclear Magnetic Resonance (NMR) Spectroscopy. 1H and 13C spectra were recorded at 25 °C, in D2O, on a Varian (Darmstadt, Germany) Unity-plus 300 spectrometer (300 and 75 MHz nominal frequency, respectively). Lyophilization. A Labconco Centrivap Concentrator (Kansas City, MO) was applied. Software. In silico digestions were performed using the protein identification and analysis tools of the ExPASy Molecular Biology Server (http://www.expasy.ch/). LC-(ESI)MS data were processed with MasslLynx 3.2 software. Positions of lysine residues and their local protein environment were determined by the Swiss-Pdb Viewer (http://www.expasy.ch/spdbv/). RESULTS
Identification and Quantification of LysozymeBound Quinoxaline 6 and Glucosepane 5 by LC(ESI)MS. Reactive R-dicarbonyl intermediates such as 2 and 4 (Figure 1) usually cannot be detected in native form in complex reaction mixtures but only as stable derivatives. The most frequently employed trapping reagents are o-phenylenediamine (OPD) (26-28) and aminoguanidine (AG) (29); the relative reactivity of these two agents has recently been compared (30). As preliminary experiments have shown, the trapping capability of AG toward the dideoxyosone 4 is much lower than that of OPD which forms N6-(2,3-dihydroxy-4-quinoxalin-2ylbutyl)-L-lysinate (6) (Scheme 1) in high yield. Compound 6 thus turned out to be the most suitable derivative for identification and quantification of 4. The crosslink glucosepane 5, which so far is the only known followup product of 4 (23, 24), was also monitored. The LC-
622 Bioconjugate Chem., Vol. 14, No. 3, 2003
Biemel and Lederer
Table 2. Peptides Obtained by Endoproteinase Glu C Digestion of Lysozyme Incubated with D-Glucose (100 mM) and OPD (50 mM) GluC peptidea
position
sequencea
calculated m/zb
observed m/zb
G1 G1* G2 G2+3 G2+3* G3 G3* G4 G4+5 G7 G8 G10
1-7 1-7 8-18 8-35 8-35 19-35 19-35 36-48 36-52 67-87 88-101 120-129
KVFGRCE K*VFGRCE LAAAMKRHGLD LAAAMKRHGLDNYRGYSLGNWVCAAKFE LAAAMKRHGLDNYRGYSLGNWVCAAK*FE NYRGYSLGNWVCAAKFE NYRGYSLGNWVCAAK*FE SNFNTQATNRNTD SNFNTQATNRNTDGSTD GRTPGSRNLCNIPCSALLSSD ITASVNCAKKIVSD VQAWIRGCRL
895.5 1111.5 1182.6 3198.6 3414.6 2035.0 2251.0 1482.7 1842.8 2275.1 1505.8 1258.7
895.4 1111.4 1182.5 3198.4 3414.4 2034.7 2251.8 1482.4 1842.8 2275.0 1505.6 1258.5
modification site K1 K13/ K33 K33
a Peptides modified with the quinoxaline 6 are marked with an asterisk. b Cysteine (C) moieties are carbamidomethylated. This derivatization has to be taken into account regarding m/z values of peptides containing C.
Table 3. Peptides Obtained by Tryptic Digestion of Lysozyme Incubated with D-Glucose (100 mM) and OPD (50 mM) tryptic peptidea
position
sequencea
calculated m/zb
observed m/zb
T1+2 T1+2* T2 T3 T3+4 T3+4* T5 T6 T6+7* T7 T8 T9 T10 T11 T11+12* T12+13 T12+13* T13 T15+16 T15+16* T16 T17+18
1-5 1-5 2-5 6-13 6-14 6-14 15-21 22-33 22-45 34-45 46-61 62-68 69-73 74-96 74-97 97-112 97-112 98-112 115-125 115-125 117-125 126-129
KVFGR K*VFGR VFGR CELAAAMK CELAAAMKR CELAAAMK*R HGLDNYR GYSLGNWVCAAK GYSLGNWVCAAK*FESNFNTQATNR FESNFNTQATNR NTDGSTDYGILQINSR WWCNDGR TPGSR NLCNIPCSALLSSDITASVNCAK NLCNIPCSALLSSDITASVNCAK*K KLVSDGNGMNAWVAWR K*LVSDGNGMNAWVAWR LVSDGNGMNAWVAWR CKGTDVQAWIR CK*GTDVQAWIR GTDVQAWIR GCRI
606.4 822.5 478.3 893.4 1049.5 1265.6 874.4 1325.6 2951.3 1428.6 1753.8 993.4 517.3 2508.2 2852.4 1803.9 2020.0 1675.8 1333.7 1549.8 1045.5 505.2
606.2 822.3 478.3 893.3 1049.4 1265.4 874.3 1325.4 2951.1 1428.4 1753.8 993.3 517.3 2507.9 2852.1 1803.8 2020.0 1675.8 1333.4 1549.6 1045.4 505.2
modification site K1
K13 K33
K96 K97 K116
a Peptides modified with the quinoxaline 6 are marked with an asterisk. b Cysteine (C) moieties are carbamidomethylated. This derivatization has to be taken into account regarding m/z values of peptides containing C.
(ESI)MS system was calibrated with independently synthesized stable-isotope-labeled reference compounds in the range of 1.8-5480.0 µg of 5/L (ISTD 281.8 µg of 5-13C6/L) and 2.8-8888.0 µg of 6/L (ISTD 533.8 µg of 6-13C6/L), respectively. The linear calibration graphs are described by the equations area ) (-53 ( 294) + (1041 ( 4)L/µg × c(5) and area ) (-1950 ( 5319) + (755 ( 29)L/µg × c(6), the values in brackets representing means ( confidence intervals (P ) 95%). Limits of detection 1.2 or 26.1 µg/L, and limits of quantitation 1.8 or 39.0 µg/L for 5 or 6, were calculated according to the recommendations of the Deutsche Forschungsgemeinschaft (DFG) (31). With this ISTD method, glucosepane 5 and quinoxaline 6 were unequivocally identified and quantified in the lysozyme incubations by LC-(ESI)MS; analyses were performed directly from the total enzymatic digests. Since lysozyme has an N-terminal lysine (see Tables 2-4), the respective R-NH2 group may also be involved in the formation of a dideoxyosone which would be a regioisomer of 4. To test this hypothesis, N2-(2,3-dihydroxy-4-quinoxalin-2-ylbutyl)-L-lysinate was independently synthesized from N-t-Boc-L-lysine and the chromatographic and spectroscopic data established (not shown). It could be clearly shown that the hydrolysates contained only minute amounts of the respective quinoxaline derivative. The N-terminal amino group thence is not an important glycation site. These findings agree
well with those reported by Tagami et al. who found the lysine NR in lysozyme not to be fructosylated (32). Figure 2 shows the quantitative results for 8-week incubations of lysozyme (37 °C, 0.7 mM) with 100 mM glucose in the presence of various OPD concentrations. Without OPD, the protein derivatization by glucosepane 5 reaches a level of 1.6 nmol/mg. As expected, the amount of 5 is reduced in the presence of OPD which traps the crosslink precursor 4. Surprisingly, formation of 5 is already suppressed by 85% at 5 mM OPD while the concentration of quinoxaline 6 remains still rather low. The value for 6 increases dramatically at 20 mM OPD (18-fold compared to 5 mM OPD) and reaches a ‘maximum’ at 50 mM OPD; the concentration of 20.5 nmol/mg corresponds to an overall lysine derivatization quota of 4.9%. To test whether OPD has a catalytic effect on the formation of the dideoxyosone 4, which might be expected due to the curve progression for 6, NR-t-Boc-L-lysine and 100 mM glucose were incubated in the presence of 5, 20, or 50 mM OPD; the concentration of lysine residues was similar to that provided by the reaction with lysozyme. In this case, the amounts determined for 6 are almost identical for all OPD concentrations. Hence, formation of 4 was proven not to be significantly catalyzed by OPD which acts only as a trapping reagent. It can hardly be determined whether a true maximum is reached at 50 mM OPD (Figure 2) since the OPD concentration cannot
Localization of AGE Precursor on Protein
Bioconjugate Chem., Vol. 14, No. 3, 2003 623
Table 4. Peptides Obtained by Chymotryptic Digestion of Lysozyme Incubated with D-Glucose (100 mM) and OPD (50 mM) chymotryptic peptidea
position
sequencea
calculated m/zb
observed m/zb
C1 C1* C2 C3 C4+5 C4+5* C6 C7+8 C9 C9* C10 C11 C12+13+14 C13+14 C15 C16 C16+17 C18a C18b C18b+19 C18b+19* C18b+19** C19 C20 C21 C21*
1-3 1-3 4-8 9-12 13-20 13-20 21-23 24-28 29-34 29-34 35-38 39-53 54-63 57-63 64-75 76-83 76-84 85-93 94-105 94-108 94-108 94-108 106-108 109-111 112-123 112-123
KVF K*VF GRCEL AAAM KRHGLDNY K*RHGLDNY RGY SLGNW VCAAKF VCAAK*F ESNF NTQATNRNTDGSTDY GILQINSSRWW QINSSRWW CNDGRTPGSRNL CNIPCSAL CNIPCSALL SSDITASVN CAKKIVSDGNGM CAKKIVSDGNGMNAW CAK*KIVSDGNGMNAW CAK*K*IVSDGNGMNAW NAW VAW RNRCKGTDVQAW RNRCK*GTDVQAW
393.3 609.3 634.3 363.2 1002.5 1218.6 395.2 576.3 695.4 911.4 496.2 1657.7 1272.7 989.5 1346.6 934.4 1047.5 893.4 1279.6 1650.8 1866.8 2082.9 390.2 375.2 1490.7 1706.8
393.2 609.2 634.2 363.2 1002.4 1218.4 395.2 576.2 695.2 911.4 496.3 1657.5 1272.5 989.5 1346.4 934.3 1047.4 893.3 1279.4 1650.7 1866.6 2082.7 390.2 375.2 1490.5 1706.8
modification site K1
K13
K33
K96/K97 K96+K97
K116
a
Peptides modified with the quinoxaline 6 are marked with an asterisk. b Cysteine (C) moieties are carbamidomethylated. This derivatization has to be taken into account regarding m/z values of peptides containing C.
Figure 2. Effect of OPD concentration on glucosepane 5 suppression and quinoxaline 6 formation. Glucosepane 5 and quinoxaline 6 were quantified in incubations of lysozyme (10 g/L) with glucose (100 mM) and OPD (0, 5, 20, or 50 mM) after total enzymatic hydrolysis of the protein. Whereas formation of 5 is effectively suppressed (85%) at 5 mM OPD, the dideoxyosone 4 requires higher OPD concentrations for an efficient trapping in form of quinoxaline 6.
be raised much higher due to its limited solubility under the given conditions. Nevertheless, at 50 mM OPD, no glucosepane 5 is detected any more, indicating almost complete trapping of its precursor 4. Hence, the following quantitative evaluations are based on reactions in the presence of 50 mM OPD. Localization and Quantitative Evaluation of Aminoketose 1 and Quinoxaline 6 by LC-(ESI)MS Peptide Mapping. To obtain information about the sitespecific modification of the lysine moieties, lysozyme incubated with 100 mM glucose and 50 mM OPD was reduced with dithiothreitol, carbamidomethylated, and
Figure 3. Endoproteinase Glu C peptide mapping. Lysozyme, incubated with glucose in the presence of 50 mM OPD for eight weeks, was digested with endoproteinase Glu C. TIC as well as UV traces at 220 and 318 nm of the LC-(ESI)MS analysis are displayed. In the 220 nm chromatogram, identified peptides are labeled with G and the corresponding numbers refer to the fragments and sequences listed in Table 2. Modified peptides bearing the quinoxaline 6 appear in the 318 nm trace and are marked with an asterisk.
partially digested by endoproteinase Glu C, trypsin, or chymotrypsin. Endoproteinase Glu C cleaves C-terminal E and D peptide bonds in phosphate-buffered medium. However, scission of D-X is less favored than that of E-X which thus yields the major fragments (33). In Table 2, the identified peptides are listed; the total ion current (TIC) and the UV traces at 220 and 318 nm from the respective LC-(ESI)MS run are displayed in Figure 3. Peptides modified with the quinoxaline 6 (marked with an asterisk) are easily detected in the 318 nm trace; this wavelength represents the longest-wavelength absorption band of the quinoxaline derivative 6. The identity of both native and modified peptides was unequivocally established by comparing the m/z values of the quasimolecular ions [M + H]+ with those obtained by in silico digestion
624 Bioconjugate Chem., Vol. 14, No. 3, 2003
Figure 4. ESI+ mass spectrum of G1*. The spectrum shows the quasimolecular ions [M + H]+ at m/z 1111.4 and [M + 2H]2+ at m/z 556.3. The fragment ion at m/z 907.3 results from the loss of H2O and 3-quinoxalin-2-ylprop-1-en-1-ol from [M + H]+ (cf. Figure 5B).
Figure 5. ESI+ mass spectra of 6-13C6, 6-13C1, and 6. The ESI+ fragmentation pattern of the quinoxaline 6 was investigated by analyzing the mass spectra of 6 (C) and its isotopomers 6-13C6 (A) and 6-13C1 (B); b and C in the structural formulas mark 13C atoms. From the relative fragment ion mass shifts of the isotopomers, it can clearly be deduced how many C atoms from the native carbohydrate backbone are eliminated in the course of the respective neutral loss. The fragmentation pathways thus could be established.
of lysozyme with the ExPASy Molecular Biology Server tools; this procedure was followed for all other investigations detailed below. In the 220 nm trace (cf. Figure 3), the only signals of sufficient intensity to allow for a detection of the respective species modified by 6 are those for G1, G2+3, G3, and G10. Only G1* and, almost at the limit of detection, G2+3* and G3* could thus be identified (G10 contains no lysine residue). The ESI+ mass spectrum of G1* in Figure 4 shows [M + H]+ at m/z 1111.4, [M + 2H]2+ at m/z 556.3, and a daughter ion of m/z 1111.4 at m/z 907.3. This loss of 204 Da can be explained by fragmentation of the quinoxaline derivative and is characteristic for this modification. As detailed experiments on the ESI+ fragmentation of native 6 and two 13Cisotopomers have shown (Figure 5), the 204 Da loss results from elimination of H2O and 3-quinoxalin-2ylprop-1-en-1-ol yielding a formiminium cation (Figure 5B). Analyses of various mass spectra of modified peptides have shown that the observability of this fragmen-
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Figure 6. Trypsin peptide mapping. Lysozyme, incubated with glucose in the presence of 50 mM OPD for eight weeks, was digested with trypsin. TIC as well as UV traces at 220 and 318 nm of the LC-(ESI)MS analysis are displayed. In the 220 nm chromatogram, identified peptides are labeled with T, and the corresponding numbers refer to the fragments and sequences listed in Table 3. Modified peptides bearing the quinoxaline 6 appear in the 318 nm trace and are marked with an asterisk.
tation unfortunately seems to depend on the nature of the peptide to which the quinoxaline is linked, i.e., absence of the 204 Da loss does not necessarily provide negative evidence for this derivatization. Especially larger peptides primarily show conventional peptide fragmentation (a, b, y-series (34)) instead of the specific loss of 204 Da in collision-induced dissociation. Anyway, with endoproteinase Glu C digestion only modifications of the lysine residues K1, K13, and K33 can be detected (see Table 2 and Figure 3). Furthermore, the very low intensities of the signals for G2+3* and G3* prevent a reasonable quantitative evaluation of the respective glycation sites, and thus solely G1* can be used for this purpose. Endoproteinase Glu C digestion thus is unsuitable for assessing all lysine glycation sites by LC-(ESI)MS peptide mapping. In the next step, trypsin was used for partial enzymatic hydrolysis of glycated lysozyme. This enzyme cleaves C-terminal K and R bonds, theoretically resulting in the formation of 18 peptide fragments. However, as can be taken from Table 3, the elimination of single amino acids could not be monitored because they show no retention under the given chromatographic conditions; e.g., T1, representing only K, was not detected. Contrary to the endoproteinase Glu C digestion, all lysine glycation sites (K1, K13, K33, K96, K97, and K116) can be clearly monitored. As already reported by Smales et al. (35) for fructosylated lysozyme (Amadori-lysozyme), trypsin does not cleave at the modified positions. This finding is confirmed by our results which show that lysine moieties bearing the quinoxaline derivative 6 are not recognized as cleavage sites by trypsin. Hence, K-modified lysozyme peptides are formed only from tryptic cleavage at R-X, as outlined in the 318 nm UV trace of Figure 6 and thus allow for a highly specific detection. In case of the adjacent lysine moieties K96 and K97, peptides were found which point to a derivatization at either K96 or K97. Detection of T11+12* proves modification at K96 because the protein chain is cleaved at K97 which thus cannot be derivatized; the reverse is true for T12+13*. A parallel modification of both positions would result in a T11-13** fragment for which no signal above the limit of detection was observed. Formation of K96/K97** thence cannot be a major glycation pathway. Although the intensities of the T* peptides would allow for a site-
Localization of AGE Precursor on Protein
Bioconjugate Chem., Vol. 14, No. 3, 2003 625 Table 5. Derivatization Quota of Lysine Moieties Determined by Chymotrypsin Peptide Mapping of Lysozyme Incubated with D-Glucose (100 mM) and OPD (50 mM) for Eight Weeksa peptide
lysine moiety
6 [%]
1 [%]
ratio 1:6
C1 C4+5 C9 C18b+19 C18b+19 C21
K1 K13 K33 K96/K97b K96+K97c K116
5.5 3.0 7.5 8.0 1.0 5.5
23.5 14.0 12.0 22.0 3.5 9.5
4.3 4.7 1.6 2.8 3.5 1.7
a The percentages given represent means of two replicates. Chymotrypsin peptide mapping does not allow for a differentiation of the glycation sites K96 and K97. The results from the tryptic digest, however, show that the bulk of 6 is located at K96 whereas 1 is almost equally distributed between the two lysine moieties. c Both lysine moieties are modified.
b
Figure 7. Chymotrypsin peptide mapping. Lysozyme, incubated with glucose in the presence of 50 mM OPD for eight weeks, was digested with chymotrypsin. TIC as well as UV traces at 220 and 318 nm of the LC-(ESI)MS analysis are displayed. In the 220 nm chromatogram, identified peptides are labeled with C and the corresponding numbers refer to the fragments and sequences listed in Table 4. Modified peptides bearing the quinoxaline 6 appear in the 318 nm trace and are marked with an asterisk.
specific quantitative evaluation of 6, this intention is hampered by the fact that the peptide fragments bearing a certain lysine residue differ strikingly for modified and unmodified species due to the reasons stated above. K33 for instance is incorporated in T6 (positions 22-33, see Table 3) whereas K33* is incorporated in T6+7* (positions 22-45). A comparable ESI response cannot a priori be expected for such peptides. Since relative quantification relies on an almost identical molar response of modified and unmodified peptides, it is not feasible in this case. Sequencing-grade chymotrypsin cleaves C-terminal F, Y, W, and L peptide bonds; the enzyme used in this study additionally showed unspecific scission at M and N93 for lysozyme. Hence, 22 peptide fragments are expected from which C1-C21 could be identified (see Table 4 and Figure 7). As in the case of trypsin, all lysine glycation sites except for K96/K97 can be differentiated. Contrary to the tryptic digestion, unmodified and modified lysine moieties are now incorporated in identical peptide fragments, a prerequisite for a relative quantitative evaluation by (ESI)MS. However, it was questionable whether the derivatization of lysine would alter the molar ESI response of a specific peptide. Only for proteins or very large peptides, covalent attachment of a glycation product can be safely assumed to constitute such a small structural modification that the ESI response remains almost unchanged (36). To test this hypothesis, an equimolar mixture of lysine, Amadori compound 1, and quinoxaline 6 was subjected to LC-(ESI)MS analysis. It could be shown that postcolumn addition of propionic acid/2propanol to the LC eluent effects both a similar ESI response of the investigated compounds and a higher overall sensitivity. We thence assume that the presence of lysine, 1, or 6 in identical peptides will almost equalize the respective ESI responses, and we have performed a quantitative evaluation for 1 and 6 by signal integration of the quasimolecular ion traces of the native peptide and the corresponding glycoforms. To guarantee that the quantification of aminoketose 1 is not affected by the presence of the glucosylamine, the enzymatic digest was stirred at pH 5 for 30 min. It is well established that glucosylamines, contrary to aminoketoses, readily decompose under these conditions (37). The results obtained
from chymotrypsin peptide mapping are listed in Table 5; the values given were obtained by normalizing the peak area of the individual glycoform by the total area (sum of native peptide and both glycoforms). The data for K1 are confirmed by the endoproteinase Glu C digestion, quantitative evaluation of which gives 25% for the Amadori product 1 and 5% for the quinoxaline 6. As detailed above, glycation at K96 and K97 cannot be distinguished by chymotrypsin peptide mapping. The values for K96/K97 hence reflect the total derivatization of both moieties. However, from the analysis of the tryptic digest, it can be deduced with all proper caution that 6 is preferentially located at K96 (see intensities of the T11+12* and T12+13* signal in the 318 nm trace of Figure 6) whereas 1 is almost equally distributed between the two lysine sites. In contrast to the digestion with trypsin, minor amounts of a peptide proving parallel modification of K96/K97 were identified in the chymotryptic digest. The overall lysine derivatization by 6, calculated from the values in Table 5, amounts to 5.2% and is very close to the percentage determined with the ISTD method after total enzymatic hydrolysis (see above). This finding supports the accuracy of the quantitative data established by LC-(ESI)MS peptide mapping. DISCUSSION
The pivotal role of dicarbonyl intermediates in the Maillard reaction is firmly established. Most investigations so far have focused on 3-DG 2, MG, and GO. However, these intermediates exist in an equilibrium between free and loosely protein-associated form and thus can readily be detoxified by mammalian enzymes, widely distributed throughout the body to protect a large number of tissues against damage. Both the NADPHdependent aldose reductase and aldehyde reductase were shown to convert 2-oxoaldehydes into monocarbonyl derivatives; 3-DG 2 thus is transformed into 3-deoxy-Dfructose (38, 39). MG and GO are also disposed by these enzymes or the glyoxalase system (40). Up to now, almost no attention was paid to R-dicarbonyl intermediates covalently linked to the protein which are supposed to be hardly accessible for such detoxifying enzymes. Vasan et al. (22) have speculated on the role of N6-(5,6dihydroxy-2,3-dioxohexyl)lysinate (3, Amadori dione) in protein cross-linking. For in vivo Maillard processes, however, the involvement of 3 has never been proven. Investigations of Huber and Ledl (28) as well as Schoetter et al. (41) indicate that 3 is derived from disaccharides rather than from glucose. This finding is confirmed by our results which definitely establish N6-(2,3-dihydroxy5,6-dioxohexyl)-L-lysinate (4, Figure 1), trapped as qui-
626 Bioconjugate Chem., Vol. 14, No. 3, 2003
noxaline 6 (Scheme 1), to be the only prominent lysinelinked R-diketo compound formed from the reaction of glucose with lysozyme. The respective quinoxaline derivative of 3, with the same molecular mass as 6, was not detected. In general, the presence of further quantitatively important protein-bound quinoxalines should be visible in the 318 nm UV trace (cf. Figs. 4, 7, and 8). The identity of 6 was verified with independently synthesized stable-isotope-labeled reference material; formation of 4 on a protein thus was proven for the first time. As shown in Figure 2, presence of OPD in incubations of lysozyme with glucose results in a competition between arginine moieties of the protein and the trapping reagent OPD. Three out of six lysine residues in lysozyme (K33, K96, and K116) have an arginine side chain in a suitable distance for an intramolecular formation of the cross-link glucosepane 5 (R5, R21, and R112). Without OPD, about 8‰ of these arginine moieties are transformed into 5. The average lysine derivatization quota by 4 at K33, K96, and K116 however is 6-7% (see Table 5); i.e., only 1013% of 4 yield glucosepane 5. These calculations rest on the reasonable assumption that at 50 mM OPD the dideoxyosone 4 is quantitatively trapped as quinoxaline 6. The curve progression for the formation of 6 (Figure 2) and the fact that only a small amount of 4 is transformed into 5 both indicate 4 to be highly reactive and other reaction pathways to be operative. Investigations are currently in progress to identify further followup products of 4. The high OPD concentration required for an efficient trapping of 4 may in part be traced back to a hampered diffusion of the reagent in the viscous protein solution. Rather, it seems that protein-linked 4 enters faster into consecutive reactions than the free compound. This conclusion is based on the observation that in comparable incubations of NR-t-Boc-L-lysine with glucose equal amounts of 6 are formed at different OPD levels. In this case, 5 mM OPD obviously suffice to efficiently trap the dideoxyosone 4 due to its ‘extended lifetime’. The lifetime hypothesis is also supported by the fact that at 5 mM OPD the formation of glucosepane 5, being characterized by a low reaction rate, is largely suppressed (cf. Figure 2). Even at low concentration, OPD effectively interferes with this slow reaction whereas trapping the bulk of 4, which is susceptible to fast transformations, requires high OPD concentrations. To get insight into the formation of the dideoxyosone 4 at specific lysine moieties with special attention to the local protein environment, we performed LC-(ESI)MS peptide mapping employing endoproteinase Glu C, trypsin, and chymotrypsin. As already addressed in the Results section, partial digestion with endoproteinase Glu C is unsuitable for assessing glycation of all six lysine moieties in lysozyme. For qualitative purposes, trypsin peptide mapping is the most suitable of the investigated procedures. To quantitatively evaluate the derivatization of specific lysine moieties, however, chymotryptic digestion is required. When addressing a specific modification problem, as in the case of K96/K97 in lysozyme (cf. Table 5), a combined analysis of the data from the tryptic and chymotryptic digest is obligatory. The term “quantitative evaluation” instead of “quantification” was chosen because the values determined rely on the presupposition that native peptides and peptides modified by the aminoketose 1 or the quinoxaline 6 have almost identical molar ESI+ responses. The findings from LC-(ESI)MS experiments with lysine, 1, and 6 strongly support this assumption. For a proper quantification, however, it would be necessary to synthesize peptides incorporating a stable-isotope-labeled derivative (e.g. 6-13C6) of the
Biemel and Lederer
investigated glycation product to allow for stable-isotope dilution analysis. Due to the large number of lysine containing peptides monitored in the present study (see Tables 2-4), this time-consuming and expensive procedure appears not justified, especially in view of the fact that lysozyme serves only as a model to establish LC(ESI)MS peptide mapping as a valuable tool for sitespecific determination of glycation products, such as the dideoxyosone 4. The stable-isotope dilution technique should be kept in mind, however, for the quantification of specific lysine modification sites in tissue or serum proteins which may point to their pathophysiological significance, e.g., the ability to form intra- or intermolecular protein cross-links. Preferred glycation of certain lysine moieties has already been established in more complex proteins than lysozyme. Only nine of 55 -NH2 groups in human serum albumin are measurably fructosylated, with K525 bearing 33% of the total fructosylation (42). Hence, accurate quantification by a stable-isotope dilution protocol can be limited to the analysis of a few peptides. With an eight-week incubation period for the glycation of lysozyme by glucose, a steady-state concentration of the Amadori product 1 can be safely assumed (3). To compare the derivatization quota of accumulated 6 with that of its precursor 1, quantitative evaluation of the aminoketose 1 was also performed. As the data in Table 5 show, lysine fructosylation is in the range of 9.5-14.5% for K13-K116, only K1 shows a considerably elevated value (23,5%). The ratio 1:6, representing the effectivity of the respective transformation, ranges from 4.7 to 1.6. K33 and K116, as well as K96, show comparatively low ratios; the value for K96 is not explicitly given in Table 5, but is definitely below the average of 2.8 for K96/K97 as deduced from the results of the trypsin peptide mapping. Since the three lysine residues K33, K96, and K116 have an arginine side chain (R5, R21, and R112, respectively) at less than 5 Å distance, it seems likely that the guanidine group has a catalytic effect on the enolization reactions which transform 1 into 4. This interesting correlation suggests that formation of glucosepane 5 is in part an autocatalytic process and underlines the role of 5 as quantitatively most important Maillard cross-link known to date (23). LC-(ESI)MS peptide mapping in principle should also be suitable for the site-specific determination of 5. However, the low amounts formed (only about 8‰ of R5, R21, and R112 are transformed into 5) hamper unequivocal identification of the corresponding cross-linked peptides due to sensitivity problems; investigations are currently in progress to overcome this analytical obstacle. In conclusion, the present study has clearly established N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysinate (4) as a quantitatively important posttranslational protein modification, with the respective lysine derivatization quota ranging from 3 to 7.5%. Compound 4 is the first proteinlinked R-dicarbonyl moiety identified so far. The high yield and reactivity clearly point to the potential of 4 as exogenous or endogenous glycotoxin and thus to its key role in AGE formation. Since only a small amount of 4 is involved in the formation of 5, it must be concluded that the major follow-up products are yet unknown. Especially internal lysine residues of globular proteins modified by 4 may constitute significant glycotoxins, if they are exposed by gastrointestinal digestion from food proteins or by catabolic metabolism of tissue or plasma proteins. LC-(ESI)MS peptide mapping will be a very helpful tool to identify such glycation sites. Due to its carcinogenic properties, OPD cannot be used as trapping reagent for
Localization of AGE Precursor on Protein
in vivo investigations; the quinoxaline derivative 6 thus is ruled out as analytical in vivo-probe for 4. Aminoguanidine, on the other hand, transforms 4 into a triazine, and is already used in diabetes therapy. Because of the low yield of this transformation, AG is a problematical agent for detecting or even quantifying 4. We are currently testing new trapping reagents which exhibit both a good tolerance by the living organism and a high reaction rate for an efficient transformation of 4 into a stable derivative. Such compounds may at the same time represent novel antiglycation drugs. ACKNOWLEDGMENT
This work was supported by grant LE 1152/2-2 of the Deutsche Forschungsgemeinschaft. We thank Priv.-Doz. Dr. P. Fischer, Institute of Organic Chemistry, University of Stuttgart, for many helpful discussions and Prof. Dr. W. Schwack, University of Hohenheim, for the excellent working conditions at the Institute of Food Chemistry. To S. Mika, Institute of Chemistry, University of Hohenheim, we are grateful for the recording of the NMR spectra. LITERATURE CITED (1) Ledl, F., and Schleicher, E. (1990) New aspects of the Maillard reaction in foods and in the human body. Angew. Chem., Int. Ed. Engl. 29, 565-594. (2) Friedman, M. (1996) Food browning and its prevention an overview. J. Agric. Food Chem. 44, 631-653. (3) Schleicher, E., and Wieland, O. H. (1986) Kinetic analysis of glycation as a tool for assessing the half-life of proteins. Biochim. Biophys. Acta 884, 199-205. (4) Vlassara, H., Bucala, R., and Striker, L. (1994) Pathogenic effects of advanced glycosylation - biochemical, biologic, and clinical implications for diabetes and aging. Lab. Invest. 70, 138-151. (5) Grandhee, S. K., and Monnier, V. M. (1991) Mechanism of formation of the Maillard protein cross-link pentosidine: glucose, fructose, and ascorbate as pentosidine precursors. J. Biol. Chem. 266, 11649-11653. (6) Ulrich, P., and Cerami, A. (2001) Protein glycation, diabetes, and aging. Recent Prog. Horm. Res. 56, 1-21. (7) Vlassara, H., and Palace, M. R. (2002) Diabetes and advanced glycation endproducts. J. Intern. Med. 251, 87-101. (8) Miyata, T., Oda, O., Inagi, R., Iida, Y., Araki, N., Yamada, N., Horiuchi, S., Taniguchi, N., Maeda, K., and Kinoshita, T. (1993) β2-Microglobulin modified with advanced glycation end products is a major component of hemodialysis-associated amyloidosis. J. Clin. Invest. 92, 1243-1252. (9) Vlassara, H., Striker, L. J., Teichberg, S., Fuh, H., Li, Y. M., and Steffes, M. (1994) Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc. Natl. Acad. Sci. U.S.A. 91, 11704-11708. (10) Yang, C. W., Vlassara, H., Peten, E. P., He, C. J., Striker, G. E., and Striker, L. J. (1994) Advanced glycation end products up-regulate gene expression found in diabetic glomerular disease. Proc. Natl. Acad. Sci. U.S.A. 91, 94369440. (11) Makita, Z., Bucala, R., Rayfield, E. J., Friedman, E. A., Kaufman, A. M., Korbet, S. M., Barth, R. H., Winston, J. A., Fuh, H., Manogue, K. R., Cerami, A., and Vlassara, H. (1994) Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure. Lancet 343, 1519-1522. (12) Bucala, R., Makita, Z., Vega, G., Grundy, S., Koschinsky, T., Cerami, A., and Vlassara, H. (1994) Modification of lowdensity lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc. Natl. Acad. Sci. U.S.A. 91, 9441-9445 (13) Reiser, K. M. (1998) Nonenzymatic glycation of collagen in aging and diabetes. Proc. Soc. Exp. Biol. Med. 218, 2337. (14) Nagaraj, R. H., Sell, D. R., Prabhakaram, M., Ortwerth, B. J., and Monnier, V. M. (1991) High correlation between
Bioconjugate Chem., Vol. 14, No. 3, 2003 627 pentosidine protein cross-links and pigmentation implicates ascorbate oxidation in human lens senescence and cataractogenesis. Proc. Natl. Acad. Sci. U.S.A. 88, 10257-10261. (15) Koschinsky, T., He, C. J., Mitsuhashi, T., Bucala, R., Liu, C., Buenting, C., Heitmann, K., and Vlassara, H. (1997) Orally absorbed reactive glycation products (glycotoxins) an environmental risk factor in diabetic nephropathy. Proc. Natl. Acad. Sci. U.S.A. 94, 6474-6479. (16) Glomb, M. A., and Monnier, V. M. (1995) Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the maillard reaction. J. Biol. Chem. 270, 10017-10026. (17) Wells-Knecht, K. J., Brinkmann, E., and Baynes, J. W. (1995) Characterization of an imidazolium salt formed from glyoxal and NR-hippuryllysine - a model for maillard reaction cross-links in proteins. J. Org. Chem. 60, 6246-6247. (18) Ahmed, M. U., Brinkmann-Frye, E., Degenhardt, T. P., Thorpe, S. R., and Baynes, J. W. (1997) N-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem. J. 324, 565-570. (19) Nagaraj, R. H., Shipanova, I. N., and Faust, F. M. (1996) Protein cross-linking by the maillard reaction - isolation, characterization, and in vivo detection of a lysine-lysine crosslink derived from methylglyoxal. J. Biol. Chem. 271, 1933819345. (20) Niwa, T., Katsuzaki, T., Ishizaki, Y., Hayase, F., Miyazaki, T., Uematsu, T., Tatemichi, N., and Takei, Y. (1997) Imidazolone, a novel advanced glycation end product, is present at high levels in kidneys of rats with streptozotocin-induced diabetes. FEBS Lett. 407, 297-302. (21) Portero-Otin, M., Nagaraj, R. H., and Monnier, V. M. (1995) Chromatographic evidence for pyrraline formation during protein glycation in vitro and in vivo. Biochim. Biophys. Acta 1247, 74-80. (22) Vasan, S., Zhang, X., Kapurniotu, A., Bernhagen, J., Teichberg, S., Basgen, J., Wagle, D., Shih, D., Terlecky, I., Bucala, R., Cerami, A., Egan, J., and Ulrich, P. (1996) An agent cleaving glucose-derived protein cross-links in vitro and in vivo. Nature 382, 275-278. (23) Biemel, K. M., Friedl, D. A., and Lederer, M. O. (2002) Identification and quantification of major Maillard cross-links in human serum albumin and lens protein: evidence for glucosepane as the dominant compound. J. Biol. Chem. 277, 24907-24915. (24) Biemel, K. M., Conrad, J., and Lederer, M. O. (2002) Unexpected carbonyl mobility in aminoketoses: the key to major Maillard cross-links. Angew. Chem., Int. Ed. 41, 801804. (25) Glomb, M. A., and Pfahler, C. (2001) Amides are novel protein modifications formed by physiological sugars. J. Biol. Chem. 276, 41638-41647. (26) Beck, J., Ledl, F., and Severin, T. (1988) Formation of 1-deoxy-D-erythro-2,3-hexodiulose from Amadori compounds. Carbohydr. Res. 177, 240-243. (27) Beck, J., Ledl, F., and Severin, T. (1989) Formation of glucosyl deoxyosones from Amadori compounds of maltose. Z. Lebensm. Unters. Forsch. 188, 118-121. (28) Huber, B., and Ledl, F. (1990) Formation of 1-amino-1,4dideoxy-2,3-hexodiuloses and 2-aminoacetylfurans in the Maillard reaction. Carbohydr. Res. 204, 215-220 (29) Hirsch, J., Petrakova, E., and Feather, M. S. (1992) The reaction of some dicarbonyl sugars with aminoguanidine. Carbohydr. Res. 232, 125-130. (30) Glomb, M. A., and Tschirnich, R. (2001) Detection of R-dicarbonyl compounds in Maillard reaction systems and in vivo. J. Agric. Food Chem. 49, 5543-5550. (31) Walter, H.-F., Holtz, K.-H., Frehse, H., Gorbach, S. G., and Thier, H.-P. (1991) Ableitung von Nachweis- und Bestimmungsgrenze nach dem Eichgeradenverfahren. Ru¨ ckstandsanalytik von Pflanzenschutzmitteln - Mitteilung VI der Senatskommission fu¨ r Pflanzenschutz-, Pflanzenbehandlungs-, und Vorratsschutzmittel - Methodensammlung der Arbeitsgruppe ‘Analytik’ (Senatskommission der Deutschen Forschungsgemeinschaft, Ed.) Chapter XI-A, pp 1-22, VCH, Weinheim.
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Biemel and Lederer of aminophospholipids formed in vivo. Anal. Biochem. 272, 48-55. (38) Feather, M. S., Flynn, T. G., Munro, K. A., Kubiseski, T. J., and Walton, D. J. (1995) Catalysis of reduction of carbohydrate 2-oxoaldehydes (osones) by mammalian aldose reductase and aldehyde reductase. Biochim. Biophys. Acta Gen. Subj. 1244, 10-16. (39) Hayase, F., Liang, Z. Q., Suzuki, Y., Chuyen, N. V., Shinoda, T., and Kato, H. (1991) Enzymatic metabolism of 3-deoxyglucosone, a Maillard intermediate. Amino Acids 1, 307-318. (40) Thornalley, P. J. (1994) Methylglyoxal, glyoxalases and the development of diabetic complications. Amino Acids 6, 1523. (41) Schoetter, C., Pischetsrieder, M., Lerche, H., and Severin, T. (1994) Formation of aminoreductones from maltose. Tetrahedron Lett. 35, 7369-7370. (42) Iberg, N., and Flu¨ckiger, R. (1986) Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites. J. Biol. Chem. 261, 13542-13545.
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