Proteomic Analysis of the Site Specificity of Glycation and Carboxymethylation of Ribonuclease Jonathan W. C. Brock,† Davinia J. S. Hinton,‡ William E. Cotham,† Thomas O. Metz,† Suzanne R. Thorpe,† John W. Baynes,† and Jennifer M. Ames*,†,§ Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, and Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading, RG6 6AP, United Kingdom Received February 28, 2003
Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation endproduct (AGE), N-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 °C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors. Keywords: ribonuclease • glycation • N-(carboxymethyl)lysine • advanced glycation end-products • fructoselysine • Maillard reaction • liquid chromatography-mass spectrometry
Introduction Reactions between reducing sugars and primary amino groups in proteins occur naturally in the human body and are accelerated during hyperglycemia in diabetes. Increased chemical modification of proteins by glucose is implicated in the pathogenesis of long-term diabetic complications, including vascular and renal disease and blindness. The adduction of sugar to protein is described as glycation and occurs through the formation of a Schiff base intermediate followed by an Amadori rearrangement to give the ketoamine adduct. When glucose is the reducing sugar, the Amadori rearrangement * To whom correspondence should be addressed. Phone: +44 118 931 8730. Fax: +44 118 931 0080. E-mail:
[email protected]. † Department of Chemistry and Biochemistry, University of South Carolina. ‡ Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading. § Permanent address: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading, RG6 6AP, United Kingdom.
506
Journal of Proteome Research 2003, 2, 506-513
Published on Web 07/01/2003
product (ARP) is known as fructoselysine (FL). Further rearrangment, fragmentation and oxidation reactions of FL lead to the formation of advanced glycation end-products (AGEs), such as N-(carboxymethyl)lysine (CML). AGEs are irreversible chemical modifications and cross-links in proteins, and CML is quantitatively one of the major AGEs detected in tissue proteins.1 CML has been identified in human lens proteins and tissue collagens,2 and the concentration in human lens proteins3 and human skin collagen4 increases significantly with age and in the presence of diabetes.5 The concentration of CML in skin collagen is also correlated with the severity of diabetic complications, including renal, retinal, and vascular disease.6 Although numerous AGEs have been described,7-9 the relationship between sites of glycation and AGE formation has not been studied, nor is there any understanding of the structural features in protein that catalyze AGE formation. Determination of the distribution of protein modifications, including AGEs, is important because, overall, the degree of 10.1021/pr0340173 CCC: $25.00
2003 American Chemical Society
Ribonuclease Glycation and Carboxymethylation Scheme 1. Sequence of Amino Acids,13 Sites of Trypsin Cleavage, |,14 and Major Sites of Lysine Modification (*) of RNase
modification might be relatively low, but a high proportion of modification at one or two reactive amino acid residues would exacerbate effects of AGEs on protein functionality in aging and diabetes. Knowledge of the specificity of AGE formation may also have implications for the design of AGE inhibitors, e.g., the merits of chelating activity or anionic versus cationic inhibitors. There are two routes to CML in glucose-protein incubations, the first through glyoxal formed by autoxidation of glucose or the Schiff base.10,11 The second route is from FL, by oxidative cleavage between C2 and C3 of the sugar residue.2 The rate of formation of CML from FL depends on the phosphate concentration in vitro and occurs via a free radical mechanism.2 Cleavage of the Schiff base and the Amadori compound were identified as major routes to CML in a glucose-lysine model system.10,12 In glucose-protein systems, however, arginine will compete for the glyoxal formed by the various routes described above because glyoxal has a high reactivity toward arginine.11 Thus, the pathway to formation of CML during glycation of proteins remains in some doubt. In this paper we use the model protein ribonuclease A (RNase), to assess the specificity and pathways of formation of CML in proteins. RNase is a small (∼13.7 kDa), wellcharacterized protein containing 10 epsilon amino groups from lysine residues and one alfa amino group.13 The amino acid sequence of RNase is shown in Scheme 1. In studies on glycation of RNase, the alfa amino group of K1 was identified as the primary site of Schiff base formation (80-90% of the total). In contrast, the epsilon amino groups of K41 and K37 accounted for ∼38 and 29%, respectively of the ketoamine adducts.15 The presence of vicinal or neighboring acidic or basic residues or the binding of phosphate enhanced the reactivity of these three amino acid residues.16 We describe here the application of ESI-LC-MS to the proteomic analysis of tryptic digests of RNase modified by glucose and glyoxal. We use a semiquantitative approach to confirm earlier work identifying K1, K7, K37, and K41 as the main sites of FL formation and report for the first time that K7, K37, and K41 are also the main sites of CML formation. Furthermore, we provide evidence that FL is the main precursor of CML in the glucose-RNase (GR) incubations and compare sites of CML formation from glyoxal in the glyoxal-RNase (GOR) incubations.
Experimental Section Reagents. The following reagents were purchased from Sigma (St Louis, MO): D-(+)-glucose (ACS grade), ribonuclease A (RNase type II-A), glyoxal, trypsin (sequencing grade). Modification of RNase by Glucose or Glyoxal. Solutions were prepared in 0.2 µm filtered deionized water. RNase (13.7 mg, 1 µmol) was dissolved in 1 mL of a solution of glucose (0.4 M) or glyoxal (1, 2 or 5 mM) in phosphate buffer (0.2 M, pH 7.4) in a plastic Eppendorf tube (1.5 mL). Solutions were divided into three aliquots and one drop of toluene was added, to maintain sterile conditions, followed by aerobic incubation
research articles (that is, under air) at 37 °C for 3, 7, and 14 days (glucose) or 1, 3, and 7 days (glyoxal). Incubations were terminated by storage at -20 °C. In addition, pre-glycated RNase was prepared by incubating RNase with glucose under anaerobic conditions (that is, 1 mM diethylenetriaminepentaacetic acid (DTPA) and flushing with nitrogen for several minutes before incubation). Residual glucose and low molecular mass reaction products were removed from the incubations by ultrafiltration at 5000 ×g, using ultrafree-MC centrifugal filter devices with a nominal molecular mass cutoff of 5 kDa (Millipore, Bedford, MA), prerinsed with two 500 µL aliquots of water. The retentate (containing the protein) was washed three times by addition of water (200 µL) and centrifugation at 5000 ×g, then resuspended in 1.0 mL 0.2 M phosphate buffer and finally divided between two Eppendorf tubes. Copper sulfate (100 µM) was added to one tube and incubation of both tubes was continued for 7 days under aerobic conditions. All incubations were performed in duplicate except those using 1 and 2 mM glyoxal, which were performed only once. Tryptic Digestion. Low molecular mass components were removed from aliquots of reaction mixture (100 µL containing 1 mg RNase) by ultrafiltration, and washed as described above. The resulting retentate (ca. 50 µL) was transferred to a plastic Eppendorf tube with 250 µL 0.1 M, pH 7.2 3-(N-morpholino)propanesulfonic acid (MOPS) containing 8 M urea and 1 mM ethylenediaminetetraacetic acid (EDTA) and vortexed for 10 s. Dithiothreitol (DTT, 22 µmol), dissolved in 1 mL MOPS-ureaEDTA buffer (50 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 (25 µL) was dissolved in methanol (25 µL), mixed with water (50 µL) and a 6 µL aliquot (6 µmol) was added to the reduced protein solution, followed by incubation in the dark at room temperature for 1 h. 2-Mercaptoethanol (40 µL, 93 µmol) was mixed with water (40 µL) and 6.5 µL was added to the derivatized protein solution, with vortexing for 10 s. The solution was centrifuged (5000 ×g) in a pre-rinsed ultrafiltration tube and washed with water (3 × 200 µL) by centrifugation at 5000 ×g. The resulting retentate was transferred to a plastic Eppendorf tube and diluted to 62.5 µL with water. An aqueous solution of 8 M urea and 4% ammonium bicarbonate (37.5 µL) was added. HCl (1 mM, 100 µL) was added to a vial containing 100 µg of sequencing grade trypsin and vortexed for 10 s. Trypsin solution (50 µL) was added to the protein solution, resulting in an enzyme:substrate ratio of 5:100 (m/m), followed by incubation at 37 °C for 5 h. Digestions were immediately cooled to 4 °C, divided into aliquots (20 µL) and stored at -20 °C. Liquid Chromatography-Mass Spectrometry (LC-MS) for Proteomic Analysis. Samples were fractionated on an Agilent (Palo Alto, CA) series 1100 liquid chromatograph, coupled to either a Micromass (Manchester, UK) Quattro mass spectrometer or a Micromass quadrupole-time-of-flight (Q-TOF) mass spectrometer. A 5 µm, 100 Å pore size Aquasep C18 column (15 cm, 2 mm i.d.) was used (ES Industries, West Berlin, NJ). Tryptic digests were diluted 50:50 with water and 10 µL aliquots (∼2.5 nmol; 34 µg protein) were injected using solvent A: 0.1% aqueous trifluoroacetic acid (TFA) for the Quattro or 0.1% aqueous formic acid for the Q-TOF, and solvent B: acetonitrile, with a flow rate of 0.2 mL/min. The column was equilibrated in 2% solvent B prior to injection. After 2 min, the gradient was started and increased to 50% solvent B over 38 min. The mass spectrometers were operated in electrospray ionization (ESI) positive mode, using a mass range of 200-1800 amu Journal of Proteome Research • Vol. 2, No. 5, 2003 507
research articles
Brock et al.
Table 1. Mass:Charge (m/z) Ratios of the Predicted Charged Forms of the Unmodified, FL and CML Species of the Peptides Containing K1, K7, K37, and K41
Table 2. Choice of Charged Form Affects the Apparent Distributiona of Peptides with Glycation at K7b charged form
charged form peptide
K1K7 N34K37 C40K41K61a K1glcK7 K1glc,glcK7 K1K7glcR10 K1glcK7glcR10 K1glc,glcK7glcR10 N34K37glcR39 C40K41glcK61a K1K7cmlR10 N34K37cmlR39 C40K41cmlK61a
1+
2+
unmodified species 718 360 475 238 mrc 1307 FL species 880 441 1042 522 1313 657 1475 738 ndd 819 908 455 mr 1388 CML species nd 605 805 403 mr 1336
3+
4+
nfb nf 872
nf nf 654
nf nf 438 492 546 nf 926
nf nf nf nf 410 nf 695
404 nf 891
nf nf 669
a m/z ratios take account of derivatization of the C residues by 4-vinylpyridine. b Not formed. c Outside the scanned mass range (200-1800 amu). d Not detected.
(Quattro) or 200-1500 amu (Q-TOF). Other significant operating conditions for the Quattro were: scan time, 3.75 s; source block temperature, 80 °C; desolvation temperature, 350 °C; capillary voltage, 3.11 kV. The same operating conditions were used for the Q-TOF instrument except that the desolvation temperature was 300 °C. System control and data analysis were carried out using Micromass MassLynx and BioLynx software. Each predicted peptide (unmodified and modified) was located within the Quattro chromatogram by calculating the masses of the different charged forms, reconstructing single ion chromatograms and confirming the same retention time for each charged form. The masses of the predicted charged forms of the unmodified, FL and CML species of the peptides containing K1, K7, K37, and K41 are shown in Table 1. The charge state of each peptide form was confirmed on the Q-TOF in survey mode. Semiquantitative data for the peptides were obtained in two ways. First, the amount of each peptide was expressed as a percentage of the sum of a group of related peptides (based on the sum of the peak areas of their different charged forms). Second, the relative amount (RA) of each peptide was calculated by dividing the sum of the peak areas of its different charged forms by the sum of the peak areas of the charged forms of the C-terminal peptide of RNase (H105V124) in the incubation. The latter peptide does not contain a lysine or arginine residue and was recovered in ∼98% yield in modified, compared to native, protein.
Results Analysis of LC-MS Data. Figure 1 gives an example of the location of a peptide, i.e., the glycated form of peptide CKPVNTFVHESLADVQAVCSQK (C40K41glcK61) within a Quattro total ion chromatogram (TIC, Figure 1A) for GR incubated for 14 days. The 2+, 3+, and 4+ ions are formed on ESI and the reconstructed selected ion chromatograms (SICs) for their corresponding m/z values, 1388, 926, and 695, are shown in Figure 1, parts B, C, and D, respectively. All three SICs show a single or major peak at the same retention time (26.94 min), indicating that the ions are different charged forms of the same 508
Journal of Proteome Research • Vol. 2, No. 5, 2003
glycated peptide
1+
2+
3+
4+
allc
K1K7glcR10 K1glcK7glcR10 K1glc,glcK7glcR10
78 22 0
51 42 7
42 45 13
0 0 100
49 41 10
a Peak area of each glycated peptide expressed as a percentage of the sum of the peak areas of each charged form. b Data taken from one LC-MS run of GR incubated for 14 days in phosphate buffer. c Apparent distribution calculated from the sum of the peak areas of the different charge forms.
peptide. The Quattro mass spectrum at 26.94 min is given in Figure 1E, and major ions are observed at m/z values, 1388, 926, and 695. Q-TOF mass spectra of the 3+ and 4+ ions over a narrower m/z range are shown in Figure 1, parts F and G, and confirm their predicted charge states. (The 2+ ion was too weak for charge state confirmation.) Due to the natural abundance of the 13C isotope, isotope peaks for each ion are seen at 0.33 atomic mass unit (amu) intervals for (triply charged ions), and at 0.25 amu intervals (for quadruply charged ions). Singly and doubly charged ions were observed for the smaller tryptic peptides and they gave isotope peaks at one and 0.5 amu units, respectively, confirming their charge states. The need to consider all of the charged form of the peptide when calculating amounts of each species is illustrated in Table 2, using the three different glycated forms of K7 present in GR incubated for 14 days as an example. Increasing glycation increases formation of the more highly charged forms. For example, the 1+ ion of K1glcglcK7glcR10 was not detected, whereas the same peptide was the only one giving detectable amounts of the 4+ ion. Thus, it was important to take account of all the detected charged forms of each peptide when calculating semiquantitative data. H105V124 was modified to only a small extent during incubation with glucose (about 2% was involved in run-on from T99K104, due to slight modification at K104 at 14 days). Therefore, H105V124 was used as an “internal standard” to obtain semiquantitative data on the extent of modification of the different peptide species. Ratioing the peak areas for the different charged forms of each peptide to that of H105V124 in the same LC-MS run gave relative amounts (RAs) that were dimensionless and that were related to the absolute amount of each component present. Location of Major Sites of FL and CML Formation in GR. All ten K residues became modified to FL on incubating RNase with glucose in phosphate buffer but the extent of modification at each residue varied greatly. The major sites of FL and CML formation within the protein are shown in Scheme 1. Initial rates of glycation (formation of FL at 3 days) identified K41 and K7 as the primary sites of modification of RNase, yielding C40K41glcK61 and K1K7glcR10 (Figure 2), ∼53 and 30%, respectively (Table 3). RAs of these glycated forms increased at 7 days, followed by a decrease at 14 days (Figure 2), possibly due to cross-linking.17 The proportion of overall modification at K7 (peptide K1K7glcR10) and K41 (peptide C40K41glcK61) appears to decrease after 3 days, but this results from the increase in glycation at K1 and K61, respectively, as well as at K37 (Table 3). This is most apparent for K7, which appears first (at 3 days) mainly as K1K7glcR10, together with a small amount of K1glcK7glcR10. Peptide K1glc,glcK7glcR10 was first detected at 7 days and, together with K1glcK7glcR10, accounted for
Ribonuclease Glycation and Carboxymethylation
research articles
Figure 1. ESI-LC-MS data showing the location and identification of the peptide C40K41glcK61. GR was incubated aerobically for 14 days and the tryptic digest (see the Experimental Section) containing ∼2.5 nmol protein (10 µL) was fractionated by reverse phase LC using a gradient from 0.1% aqueous modifier (TFA for Quattro or formate for Q-TOF) to acetonitrile (see the Experimental Section). A-E. Data obtained using the Quattro MS. A. TIC showing the chromatography peak containing the peptide C40K41glcK61. B. SIC at m/z 1388, showing the retention time of the 2+ ion of C40K41glcK61. C. SIC at m/z 926, showing the retention time of the 3+ ion of C40K41glcK61. D. SIC at m/z 695, showing the retention time of the 4+ ion of C40K41glcK61. E. Mass spectrum of the chromatography peak eluting at 26.96 min in B-D. F and G. Data obtained using the Q-TOF mass spectrometer. F. Mass spectrum of the 3+ ion showing that the mass difference between isotopic peaks is ∼0.33 amu. G. Mass spectrum of the 4+ ion showing that the mass difference between isotopic peaks is ∼0.25 amu.
increasing proportions of glycation at K7 up to 14 days (Table 3). The sequence of formation of the different K1 and K7 modifications is outlined in Scheme 2. At 14 days, modification at K41 accounted for ∼39% of the total FL in the protein, while several K residues contributed only 1-2% of the total amount. Because K1, K7, K37, and K41 were the most highly modified residues, each accounting for >5% of the total FL, the extent of their modification was monitored during this study. K7, K37, and K41 were also the major sites of CML formation in phosphate after 3 days (Figure 3), although trace levels ( K7 > K37, in agreement with the order of glycation (K41 > K7 > K37). Profile of CML Species in Pre-Glycated GR Incubations. To assess the role of Amadori adducts (FL) as precursors of CML, RNase was pre-glycated by incubating with glucose under anaerobic conditions for 7 days followed by removal of residual glucose and low molecular mass reactions products. The resulting pre-glycated protein was then incubated in phosphate Journal of Proteome Research • Vol. 2, No. 5, 2003 509
research articles
Brock et al.
Figure 2. Kinetics of formation of glycated tryptic peptides in aerobic glucose-ribonuclease (GR) incubations. Amounts of peptides are given relative to the amount of the C-terminal peptide, H105V124, (see the Experimental Section). Data points are the mean values for duplicate incubations. Error bars represent the range.
Figure 3. Kinetics of formation of carboxymethylated tryptic peptides in aerobic glucose-ribonuclease (GR) incubations. Amounts of peptides are given relative to the amount of the C-terminal peptide, H105V124, (see the Experimental Section). Data points are the mean values for duplicate incubations. Error bars represent the range.
Table 3. Percentage Amounts of FL Species in GR Incubated in Phosphate Buffer for 3, 7, and 14 days under Aerobic Conditions, and 7 days under Anaerobic Conditions for the Preparation of Pre-Glycated RNase
Table 4. Percentage Amounts of CML Species in GR Incubated in Phosphate Buffer for 3, 7, and 14 days under Aerobic Conditions, and Washed Pre-glycated RNase Incubated for 7 days under Aerobic Conditions with 100 µM Cu2+
aerobicb
pre-glycatedc
speciesa
3d
7d
14d
7d
K1glcK7 K1glc,glcK7 K1K7glcR10 K1glcK7glcR10 K1glc,glcK7glcR10 N34K37glcR39 C40K41glcK61
4 trd 30 5 nde 8 53
4 1 28 11 1 10 45
5 1 18 17 3 17 39
3 1 18 11 2 10 55
a Total percentage amountsof glycation at K1 and K7 under aerobic conditions are, respectively, 7 and 32% (3 days), 11 and 34% (7 days), and 17 and 27% (14 days). Total percentage amountsof glycation at K1 and K7 under anaerobic conditions (pre-glycated sample) are, respectively, 11 and 24%. b Values obtained for the individual incubations were within K37, and the extent of loss of the unmodified peptide was comparable in both the aerobic and anaerobic incubations. Low levels of CML were detected at K41 during the pre-glycation stage, but no detectable increase in CML occurred during aerobic incubation in phosphate buffer alone, probably because of copper removal with the chelator during dialysis of the pre-glycated incubation or 510
Journal of Proteome Research • Vol. 2, No. 5, 2003
aerobica
pre-glycatedb
species
3d
7d
14d
7d
K1K7cmlR10 N34K37cmlR39 C40K41cmlK61
20 16 64
22 12 66
13 15 72
19 19 62
a Values obtained for the individual incubations were within
