Proteomic Analysis of Arginine Adducts on Glyoxal-modified Ribonuclease
2004; Elsevier BV; Volume: 3; Issue: 12 Linguagem: Inglês
10.1074/mcp.m400002-mcp200
ISSN1535-9484
AutoresWilliam E. Cotham, Thomas Metz, P. Lee Ferguson, Jonathan W. C. Brock, Davinia J. S. Hinton, Suzanne R. Thorpe, John Baynes, Jennifer M. Ames,
Tópico(s)Biochemical effects in animals
ResumoAccumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process. Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process. Glucose and its oxidative degradation products, including glyoxal (1Wells-Knecht K.J. Zyzak D.V. Litchfield J.E. Thorpe S.R. Baynes J.W. Mechanism of autoxidative glycosylation: Identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose..Biochemistry. 1995; 34: 3702-3709Google Scholar, 2Glomb M.A. Monnier V.M. Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction..J. Biol. Chem. 1995; 270: 10017-10026Google Scholar, 3Thornalley P.J. Langborg A. Minhas H.S. Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose..Biochem. J. 1999; 344: 109-116Google Scholar), are able to modify reactive side chains of amino acids in proteins under physiological conditions to form a diverse group of protein-bound adducts known as advanced glycation end-products (AGEs). 1The abbreviations used are: AGE, advanced glycation end-product; AG, aminoguanidine; CMA, Nω-(carboxymethyl)arginine; CML Nε-(carboxymethyl)lysine; DTPA, diethylenetriaminepentaacetic acid; G-DH, glyoxal-derived dihydroxyimidazolidine; G-DH1, Nδ-(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, Nγ-(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. 1The abbreviations used are: AGE, advanced glycation end-product; AG, aminoguanidine; CMA, Nω-(carboxymethyl)arginine; CML Nε-(carboxymethyl)lysine; DTPA, diethylenetriaminepentaacetic acid; G-DH, glyoxal-derived dihydroxyimidazolidine; G-DH1, Nδ-(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, Nγ-(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. Such reactions, known as Maillard or browning reactions, are accelerated during hyperglycemia in diabetes, and increased chemical modification of proteins by glucose is implicated in the pathogenesis of long-term diabetic complications, including vascular and renal disease and blindness.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 (4Fu M. Requena J.R. Jenkins A.J. Lyons T.J. Baynes J.W. Thorpe S.R. The advanced glycation end product, Nε-(carboxymethyl)lysine, is a product of both lipid peroxidation and glycoxidation reactions..J. Biol. Chem. 1996; 271: 9982-9986Google Scholar) and during myeloperoxidase-mediated degradation of serine at sites of inflammation (5Anderson M.M. Hazen S.L. Hsu F.F. Heinecke J.W. Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to convert hydroxy-amino acids into glycolaldehyde, 2-hydroxypropanal, and acrolein. A mechanism for the generation of highly reactive α-hydroxy and α,β-unsaturated aldehydes by phagocytes at sites of inflammation..J. Clin. Invest. 1997; 99: 424-432Google Scholar). 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 215–230 nm (6Agalou S. Karachalias N. Thornalley P.J. Tucker B. Dawnay A.B. Estimation of α-oxoaldehydes formed from the degradation of glycolytic intermediates and glucose fragmentation in blood plasma of human subjects with uraemia.in: The Maillard Reaction in Food Chemistry and Medical Science: Update for the Postgenomic Era. International Congress Series 1245, Excerpta Medica, Elsevier, Amsterdam, The Netherlands2002: 181-182Google Scholar, 7Lapolla A. Flamini R. Tonus T. Fedele D. Senesi A. Reitano R. Marotta E. Pace G. Seraglia R. Traldi P. An effective derivatization method for quantitative determination of glyoxal and methylglyoxal in plasma samples by gas chromatography/mass spectrometry..Rapid Commun. Mass Spectrom. 2003; 17: 876-878Google Scholar, 8Lapolla A. Flamini R. Dalla Vedova A. Senesi A. Reitano R. Fedele D. Basso E. Seraglia R. Traldi P. Glyoxal and methylglyoxal levels in diabetic patients: Quantitative determination by a new GC/MS method..Clin. Chem. Lab. Med. 2003; 41: 1166-1173Google Scholar) but increase to 350–470 nm in diabetic subjects (7Lapolla A. Flamini R. Tonus T. Fedele D. Senesi A. Reitano R. Marotta E. Pace G. Seraglia R. Traldi P. An effective derivatization method for quantitative determination of glyoxal and methylglyoxal in plasma samples by gas chromatography/mass spectrometry..Rapid Commun. Mass Spectrom. 2003; 17: 876-878Google Scholar, 8Lapolla A. Flamini R. Dalla Vedova A. Senesi A. Reitano R. Fedele D. Basso E. Seraglia R. Traldi P. Glyoxal and methylglyoxal levels in diabetic patients: Quantitative determination by a new GC/MS method..Clin. Chem. Lab. Med. 2003; 41: 1166-1173Google Scholar), ∼400 nm in uraemia (6Agalou S. Karachalias N. Thornalley P.J. Tucker B. Dawnay A.B. Estimation of α-oxoaldehydes formed from the degradation of glycolytic intermediates and glucose fragmentation in blood plasma of human subjects with uraemia.in: The Maillard Reaction in Food Chemistry and Medical Science: Update for the Postgenomic Era. International Congress Series 1245, Excerpta Medica, Elsevier, Amsterdam, The Netherlands2002: 181-182Google Scholar), and ∼760 nm in end-stage renal disease (6Agalou S. Karachalias N. Thornalley P.J. Tucker B. Dawnay A.B. Estimation of α-oxoaldehydes formed from the degradation of glycolytic intermediates and glucose fragmentation in blood plasma of human subjects with uraemia.in: The Maillard Reaction in Food Chemistry and Medical Science: Update for the Postgenomic Era. International Congress Series 1245, Excerpta Medica, Elsevier, Amsterdam, The Netherlands2002: 181-182Google Scholar). 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 Nε-(carboxymethyl)lysine (CML (9Ahmed M.U. Thorpe S.R. Baynes J.W. Identification of Nε-carboxymethyllysine as a degradation product of fructoselysine in glycated protein..J. Biol. Chem. 1986; 261: 4889-4894Google Scholar)) and Nω-(carboxymethyl)arginine (CMA (10Iijima K. Murata M. Takahara H. Irie S. Fujimoto D. Identification of Nω-carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen..Biochem. J. 2000; 347: 23-27Google Scholar)), glyoxal-derived dihydroxyimidazolidines (G-DHs; G-DH1 and G-DH2) and Nδ-(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 (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar)) (Fig. 1).Little is known about the relative rates of formation of CML, glyoxal-derived dihydroxyimidazolidines (G-DHs), glyoxal-derived hydroimidazolones (G-Hs) or CMA, or the specificity of modification of proteins by glyoxal. G-Hs and CMA would both be formed via G-DH1 or its isomer 5-(4,5-dihydroxy-2-imino-1-imidazolidinyl)norvaline (G-DH2; Fig. 1).In previous work (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar) 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.EXPERIMENTAL PROCEDURESMaterials—The following reagents were purchased from Sigma (St. Louis, MO): d-(+)-glucose (ACS grade), bovine RNase A (RNase type II-A, P00656), glyoxal, trypsin (sequencing grade), hydroxylamine (HA, 99%), o-phenylenediamine (OPD). CMA was a gift from R. Nagai (University of Kumamoto, Kumamoto, Japan).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 (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar).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 (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar), 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. (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar). 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 × 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 (0–10 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 × 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 (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar), 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 (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar).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 (8F—R10) and 11QHMDSSTSAASSSNYCNQMMK31 (11Q—K31). Peptides 8F—R10 and 11Q—K31 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 11Q—K31, 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.Table IHPLC retention times (min) of peptides used to estimate modifications at arginine residues on RNase incubated for up to 7 days with 1 mm glyoxalPeptide groupUnmodified peptideaUnmodified peptides were 11Q—K31 (Arg10), 40C—K61 (Arg39), and 67N—R85 (Arg85). (Trypsin gave almost no cleavage between Lys91 and Tyr92. Therefore, modifications of peptide 67N—K98 with two missed cleavage sites were monitored.)G-DH modificationG-H modificationCML modificationArg1021.022.522.5NAbNA, not applicable. CML was not detected on any of the lysine residues of peptides within this group.Arg3927.027.027.027.5cCML was detected at Lys41.Arg8523.523.523.5NAa Unmodified peptides were 11Q—K31 (Arg10), 40C—K61 (Arg39), and 67N—R85 (Arg85). (Trypsin gave almost no cleavage between Lys91 and Tyr92. Therefore, modifications of peptide 67N—K98 with two missed cleavage sites were monitored.)b NA, not applicable. CML was not detected on any of the lysine residues of peptides within this group.c CML was detected at Lys41. Open table in a new tab For the HA reactions, peptides were expressed relative to the yield of the unmodified C-terminal peptide, which served as an internal reference, correcting for differences in amount of protein injected (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar).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 100–2,000 amu. Fragmentation spectra were interpreted manually.RESULTSLys41 in the active site of RNase is the primary site of glycation and carboxymethylation of RNase (11Brock J.W. Hinton J.S. Cotham W.E. Metz T.O. Thorpe S.R. Baynes J.W. Ames J.M. Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease..J. Proteome Research. 2003; 2: 506-513Google Scholar). Although the extent of modification of this peptide by 0.4 m glucose and 1 mm glyoxal was similar, CML was identified as the primary product derived from glucose. Although the glyoxal-derived modification was not characterized, we established that glyoxal was not a significant intermediate in the formation of CML. In the present study, we analyzed the products of modification of RNase by glyoxal.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).Fig. 2Single combined spectra indicate the presence of several modified peptides in tryptic digests of the RNase-glyoxal incubations but not in tryptic digests of the RNase-glucose incubations. Tryptic digests of the incubations of RNase with (A) 5 mm glyoxal (7-day) and (B) 0.4 m glucose (14-day) were analyzed by LC-MS using the Quattro MS in full-scan mode. Prominent ions are labeled. C–L, expansions of selected regions of the spectra to emphasize differences between the samples. C–G, RNase-glyoxal incubations showing: C, 4+ ion at m/z 736 of 38D—K61 with Arg39 modified to G-DH/CMA; D, 3+ ion at m/z 968 of 8F—K31 with Arg10 modified to G-DH and 3+ ion at m/z 975 of 38D—K61 with Arg39 modified to G-H; E, 3+ ion at m/z 982 of 38D—K61 with Arg39 modified to G-DH/CMA; F, 3+ ion at m/z 1327 of 67N—K98 with Arg85 modified to G-DH/CMA; G, 2+ ion at m/z 1473 of 38D—K61 with Arg39 modified to G-DH/CMA. H–L, RNase-glucose incubations showing absence of significant ions identified in C–G.View Large Image Figure ViewerDownload (PPT)Extraction of the ion chromatogram at m/z 982 from the full-scan data revealed one major peak, eluting at 27.21 min (Fig. 3A). The spectrum of this peak contained ions at m/z 1473, 982, and 736 (Fig. 3B), corresponding to the 2+, 3+, and 4+ ions of the same unidentified peptide that was named “Unknown 1” (UK1). Q-TOF analysis established that the proposed 3+ ion was triply charged.Fig. 3An extracted ion chromatogram at m/z 982 for the 7-day RNase-5 mm glyoxal incubation contains one major peak and its spectrum contains ions that correspond to the 2+, 3+, and 4+ ions of the same peptide (UK1; m/z = ∼2942 Da).A, extracted ion chromatogram at m/z 982 for the tryptic digest of RNase incubated with 5 mm glyoxal for 7 days showing one major peak (at 27.21 min). B, spectrum of the peak at 27.21 min showing ions at m/z 1473, 982, and 736, corresponding to the 2+, 3+, and 4+ ions of peptide UK1 with a mass ∼2942 Da. Insert is the Q-TOF survey mode data for the ion at m/z 982 showing that it is triply charged.View Large Image Figure ViewerDownload (PPT)Signals corresponding to the 2+ and 4+ ions, at m/z 1473 and 736, respectively, were also located in the single combined spectrum of the glyoxal incubation but not in that for the glucose incubation (Fig. 2, C, G, H, and L). The m/z ratios of these different charged forms imply a mass of ∼2942 Da for UK1 that was detected only in the glyoxal, and not in the glucose, reactions.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 (12Ahmed N. Argirov O.K. Minhas H.S. Cordeiro C.A.A. Thornalley P.J. Assay of advanced glycation endproducts (AGEs): Surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nε-carboxymethyl-lysine- and Nε-(1-carboxyethyl)lysine-modified albumin..Biochem. J. 2002; 364: 1-14Google Scholar,13Ahmed N. Thornalley P.J. Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence..Biochem. J. 2002; 364: 15-24Google Scholar)), G-DHs (14Glomb M.A. Lang G. Isolation and characterization of glyoxal-arginine modifications..J. Agric. Food Chem. 2001; 49: 1493-1501Google Scholar), and CMA (10Iijima K. Murata M. Takahara H. Irie S. Fujimoto D. Identification of Nω-carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen..Biochem. J. 2000; 347: 23-27Google Scholar, 15Odani H. Iijima K. Nakata M. Miyata S. Kusunoki H. Yasuda Y. Hiki Y. Irie S. Maeda K. Fujimoto D. Identification of Nω-carboxymethylarginine, a new advanced glycation endproduct in serum proteins of diabetic patients: Possibility of a new marker of aging and diabetes..Biochem. Biophys. Res. Commun. 2001; 285: 1232-1236Google Scholar). The predicted mass of UK1 corresponded to the mass of the tryptic peptide with a missed cleavage C-terminal to Arg39, peptide 38D—K61, 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 38D—K61 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.Fig. 4A sequencing spectrum confirms that peptide UK1 is modified peptide 38D—K61. However, fragment ions did not permit unambiguous assignment of the site of modification. A, MS/MS spectrum of the 3+ ion of peptide 38D—K61 with Arg39 modified to G-DH/CMA (precursor ion at m/z 982). B, expanded spectrum over the range m/z range 700–1200. 1+ and 2+ ions are shown, respectively, in normal typeface and italics. C, Amino acid sequence of peptide 38D—K61 with Arg39 modified to G-DH/CMA showing the theoretical masses of the b and y ions. Located ions are shown in italics. The cone voltage was 45 eV and the collision energy was 45 V.View Large Image Figure ViewerDownload (PPT)As shown in Fig. 4, a series of singly charged y ions (y3–y15) and the doubly charged y20 ion were observed. Some b ions were also observed but were generally less intense than the y ions. Most were doubly charged (b9, b11–b19) but some singly charged ions (b6, b9, b10, b12) were also seen. The b ions provide evidence that UK1 incorporates Arg39 and a modification accounting for 58 amu. The y ions establish that the precursor ion contains the sequence of amino acids VHESLADVQAVCS, confirming that UK1 incorporates tryptic peptide 40C—K61. All the MS data presented in Figs. 3 and 4 establish the identity of peptide UK1 to be 38D—K61 with either Arg39 modified to G-DH or CMA, or Lys41 modified to CML.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 38D—K61 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 (16Snyder A.P. Interpreting Protein Mass Spectra. A Comprehensive Resource. Oxford University Press, New York2000Google Scholar) 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 (14Glomb M.A. Lang G. Isolation and characterization of glyoxal-arginine modifications..J. Agric. Food Chem. 2001; 49: 1493-1501Google Scholar). 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 an
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