Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using

Anest Iwata Co., Yokohama, Japan). In data analysis state, the acquired chromatogram was shown using TIC mode in fragment table. This LC−MS syst...
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Anal. Chem. 2009, 81, 1819–1825

Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using Liquid Chromatography-Mass Spectrometry with an N-Terminal β-Amyloid Peptide Koichi Inoue,*,† Ako Nakagawa,† Tomoaki Hino,† and Hisao Oka†,‡ Department of Physical and Analytical Chemistry, School of Pharmacy, and Graduate School of Human Ecology, Human Ecology Major, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya 463-8521, Japan Production of microregional catalytic reactive oxygen species (ROS) by metal-binding amyloid-β (Aβ) peptides mediates the neurotoxicity of Alzheimer’s disease, and inhibitors of this activity may be of therapeutic value. No current analytical methods target specific ROS inhibitors produced by metal-binding peptides. We report a screening assay for metal-catalyzed oxidation (MCO) inhibitors based on liquid chromatography-mass spectrometry (LC-MS) with a model N-terminal Aβ peptide (Aβ1-6). When subjected to MCO by Cu(II)/ascorbic acid, singly and doubly charged Aβ1-6 molecules were observed at m/z 729.2 and 364.8 and m/z 685.3 and 343.3, respectively, corresponding to a decrease in mass of 45 and 89 Da compared with the model peptide. In contrast, H2O2 did not modify the Aβ1-6 peptide. Modified peptides were characterized by a specific MCO of Aβ1-6, which contains both His and N-terminal Asp residues. LC-MS detection of the modified peptides allowed us to identify antioxidants that inhibit MCO of Aβ1-6. MCO of the model peptide was inhibited by curcumin, but not dibutylhydroxytoluene, carotene, tocopherol, estradiol or nicotine, revealing a clear difference between curcumin and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against MCO of peptides and proteins related to degenerative diseases. Metal-catalyzed oxidation (MCO) of peptides and/or proteins is mainly a site-specific process in which only a few amino acids at the metal-binding sites are preferentially oxidized. MCO gives rise to highly reactive intermediates, such as hydroxyl radicals, which damage biologic molecules and are implicated in aging and the pathogenesis of neurodegenerative disease, including Alzheimer’s disease (AD).1,2 Studies of subjects with early onset AD indicate that metabolism and modification of amyloid-β (Aβ) * To whom correspondence should be addressed. Phone and Fax: +81-52798-0982. E-mail: [email protected]. † School of Pharmacy. ‡ Graduate School of Human Ecology. (1) Varadarajan, S.; Yatin, S.; Aksenova, M.; Butterfield, D. A. J. Struct. Biol. 2000, 130, 184–208. (2) Bush, A. I. Trends Neurosci. 2003, 26, 207–214. 10.1021/ac802162n CCC: $40.75  2009 American Chemical Society Published on Web 01/27/2009

peptides are involved.3 Recent evidence also supports a role for metal ions and reactive oxygen species (ROS) in the pathogenesis of AD.4-6 The primary interaction of Aβ peptides with metal ions has been studied extensively using various analytical techniques, such as electron spin resonance, nuclear magnetic resonance, and mass spectrometry (MS).7-11 Thus, the redox reaction of metal-Aβ complexes has been investigated in an attempt to unravel its important role in the pathogenesis of AD.12-15 The brain tightly regulates metal ion homeostasis as part of the normal physiologic processes. A growing body of evidence implicates the involvement of metal ions, particularly copper (Cu), zinc (Zn), and iron (Fe) ions, in the pathogenesis of AD.2,5 Therefore, the specific modification of Aβ peptides by MCO is key to discovering new approaches toward the prevention and therapy of AD. In a recent study to search for potential biomarkers of AD, specific oxidized Aβ peptides modified by MCO were detected using liquid chromatography-mass spectrometry (LC-MS).16 Although the methionine-35 residue (Met35) in Aβ peptides is a potential target of the ROS produced by MCO,17,18 MCO of histidine residues (His) most likely precedes the oxidation of Met35 by ROS generated from Cu/ascorbic (3) Hardy, J. Trends Neurosci. 1997, 20, 154–159. (4) To ˜ugu, V.; Karafin, A.; Palumaa, P. J. Neurochem. 2008, 104, 1249–1259. (5) Smith, D. G.; Cappai, R.; Barnham, K. J. Biochim. Biophys. Acta 2007, 1768, 1976–1990. (6) Adlard, P. A.; Bush, A. I. J. Alzheimer’s Dis. 2006, 10, 145–163. (7) Dikalov, S. I.; Vitek, M. P.; Mason, R. P. Free Radical Biol. Med. 2004, 36, 340–347. (8) Hou, L.; Zagorski, M. G. J. Am. Chem. Soc. 2006, 128, 9260–9261. (9) Smith, D. P.; Smith, D. G.; Curtain, C. C.; Boas, J. F.; Pilbrow, J. R.; Ciccotosto, G. D.; Lau, T. L.; Tew, D. J.; Perez, K.; Wade, J. D.; Bush, A. I.; Drew, S. C.; Separovic, F.; Masters, C. L.; Cappai, R.; Barnham, K. J. J. Biol. Chem. 2006, 281, 15145–15154. (10) Lim, J.; Vachet, R. W. Anal. Chem. 2003, 75, 1164–1172. (11) Lim, J.; Vachet, R. W. Anal. Chem. 2004, 76, 3498–3504. (12) Jiang, D.; Men, L.; Wang, J.; Zhang, Y.; Chickenyen, S.; Wang, Y.; Zhou, F. Biochemistry 2007, 46, 9270–9282. (13) Hayashi, T.; Shishido, N.; Nakayama, K.; Nunomura, A.; Smith, M. A.; Perry, G.; Nakamura, M. Free Radical Biol. Med. 2007, 43, 1552–1559. (14) Guilloreau, L.; Combalbert, S.; Sournia-Saquet, A.; Mazarguil, H.; Faller, P. ChemBioChem 2007, 8, 1317–1325. (15) Honda, K.; Casadesus, G.; Petersen, R. B.; Perry, G.; Smith, M. A. Ann. N.Y. Acad. Sci. 2004, 1012, 179–182. (16) Inoue, K.; Garner, C.; Ackermann, B. L.; Oe, T.; Blair, I. A. Rapid Commun. Mass Spectrom. 2006, 20, 911–918. (17) Butterfield, D. A.; Kanski, J. Peptides 2002, 23, 1299–1309. (18) Butterfield, D. A.; Boyd-Kimball, D. Biochim. Biophys. Acta 2005, 1703, 149–156.

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Table 1. Sequences and Molecular Information of Amyloid-β Peptides That Include His Residues abbr.a

amino acid’s sequence

Aβ1-42 Aβ1-40 Aβ1-16 Aβ1-6

NH2- DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA -COOH NH2-1DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV40-COOH NH2-1DAEFRHDSGYEVHHQK16-COOH NH2-1DAEFRH6-COOH

a

1

(23) (24) (25)

(26) (27)

comp. form.b

theor int. MWc

C203H311N55O60S C194H295N53O58S C84H119N27O28 C33H47N11O11

4511 4327 1954 773

Abbreviated. b Composition formula. c Theoretical integer molecular weight.

acid.16,19-21 The redox activity of transition metals present in senile plaques is controlled by the His6, His13, and His14 residues of Aβ peptides.22 Therefore, modification of the His residues in Aβ peptides may be a useful indicator for detecting MCO. The Aβ peptides that contain His residues are shown in Table 1. The Aβ sequences of 1-40 and 1-42 peptides have been used as target biomarkers of Aβ aggregation and AD pathogenesis.23,24 The use of simple possible biomarkers and/or indicators is difficult, however, because their physical properties cause numerous analytical problems, such as loss during collection, transfer, or freeze/thawing through aggregation and/or binding to surfaces.25 Therefore, smaller Aβ peptides that include His, such as 1-6 (Table 1), may be simpler indicators of MCO than Aβ1-40 and Aβ1-42. In this study, an N-terminal Aβ model peptide (Aβ1-6) was used to develop an assay to screen for MCO of Aβ peptides. Among therapeutic interventions for AD, the use of antioxidants is a promising approach for slowing progression by inhibiting oxidative stress-induced damage linked to cognitive and functional decline. Therefore, the current approach is to target the initiating event in the generation of ROS based on in vitro tests. A recent review, however, indicated that only a few types of antioxidant activity, which are the basis for the development of antioxidant pharmaceutical drugs, can be differentiated.26 Another recent report indicated that Cu/ascorbic acid-mediated ROS generation occurs exclusively at specific His residues and that H2O2 oxidation forms only oxidized Met35 in Aβ peptides.16 Thus, interactions and antioxidants to target oxidized Aβ modifications with ROS generated by MCO can be differentiated depending on the residues that are modified. A screening assay to identify the specific antioxidants that can inhibit MCO of Aβ peptides is therefore in high demand. An MS-based assay could be used to identify potential therapeutic agents for the treatment of AD based on the prevention of Aβ oxidation.27 In addition, an LC-MS assay may be utilized to discover inhibitors of Aβ aggregation.27 LC-MS assays have been developed to monitor secretase inhibitors, Aβ peptide (19) (20) (21) (22)

42

Scho ¨neich, C.; Williams, T. D. Chem. Res. Toxicol. 2002, 15, 717–722. Scho ¨neich, C. Ann. N.Y. Acad. Sci. 2004, 1012, 164–170. Opazo, C.; Ruiz, F. H.; Inestrosa, N. C. Biol. Res. 2000, 33, 125–131. Nakamura, M.; Shishido, N.; Nunomura, A.; Smith, M. A.; Perry, G.; Hayashi, Y.; Nakayama, K.; Hayashi, T. Biochemistry 2007, 46, 12737– 12743. Maccioni, R. B.; Lavados, M.; Maccioni, C. B.; Mendoza-Naranjo, A. Curr. Alzheimer Res. 2004, 1, 307–314. Borroni, B.; Premi, E.; Di Luca, M.; Padovani, A. Curr. Med. Chem. 2007, 14, 1171–1178. Oe, T.; Ackermann, B. L.; Inoue, K.; Berna, M. J.; Garner, C. O.; Gelfanova, V.; Dean, R. A.; Siemers, E. R.; Holtzman, D. M.; Farlow, M. R.; Blair, I. A. Rapid Commun. Mass Spectrom. 2006, 20, 3723–3735. Ono, K.; Hamaguchi, T.; Naiki, H.; Yamada, M. Biochim. Biophys. Acta 2006, 1762, 575–586. Cheng, X.; van Breemen, R. B. Anal. Chem. 2005, 77, 7012–7015.

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metabolism, and transthyretin amyloidosis.28-30 An LC-MS assay to screen inhibitors of MCO-induced modification of Aβ peptides, however, has not been reported. Here we report the development of an LC-MS assay to screen for antioxidants that inhibit MCO of Aβ. Our approach overcomes the limitations of previous antioxidant assays and directly measures the specific MCO of Aβ peptides. In this study, curcumin was used to validate our assay because this substance has important antioxidant and anti-AD activities.31-33 We discovered that curcumin has some peculiarities not shared by other antioxidants that allows it to specifically inhibit the MCO of peptides. MATERIALS AND METHODS Materials. Synthetic Aβ1-6 (peptide purity, 98.40%; molecular weight, 773) was obtained from Sigma-Aldrich (St. Louis, MO). Copper(II) sulfate anhydrous (CuSO4), zinc(II) sulfate anhydrous (ZnSO4), iron(II) sulfate heptahydrate (FeSO4), ascorbic acid, hydrogen peroxide (H2O2), HPLC-grade water, methanol, acetonitrile, dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), 1 M Tris-HCl buffer (pH 7.5), dibutylhydroxytoluene (BHT), dopamine hydrochloride, dl-epinephrine, acetylcholine chloride, β-carotene, R-tocopherol, nicotine, and β-estradiol were obtained from Wako Chemical Co. (Osaka, Japan). Curcumin was obtained from San-Ei Gen FFI Co. (Osaka, Japan). Cholesterol was obtained from Doosan Research Laboratories (Toronto, Canada). Norepinephrine was obtained from Sigma-Aldrich (St. Louis, MO). Purified water was obtained using a Milli-Q Simplicity UV system (Millipore, Bedford, MA). Equipment. LC-MS was performed using an LCMS-2010EV system (Shimadzu Co., Kyoto, Japan) that was coupled to a quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source. LC separation was performed using a TSKGEL ODS 100V column (2.0 mm × 150 mm, 3 µm: Tosoh Co., Tokyo, Japan). LC-MS. The mobile phase consisted of 0.5% aqueous TFA (solvent A) and 0.5% TFA in methanol (solvent B). The LC linear gradient was as follows: 15% solvent B at 0 min, 40% solvent B at 30 min, 95% solvent B at 30.1 min, 95% solvent B at 35.0 min, and 15% solvent B at 35.1 min with a flow rate of 0.2 mL/min. The (28) Matthews, C. Z.; Woolf, E. J. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2008, 863, 36–45. (29) Ford, M. J.; Cantone, J. L.; Polson, C.; Toyn, J. H.; Meredith, J. E.; Drexler, D. M. J. Neurosci. Methods 2008, 168, 465–474. (30) Gupta, S.; Chhibber, M.; Sinha, S.; Surolia, A. J. Med. Chem. 2007, 50, 5589–5599. (31) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem. 2005, 280, 5892–5901. (32) Baum, L.; Ng, A. J. Alzheimer’s Dis. 2004, 6, 367–377. (33) Liu, Y.; Dargusch, R.; Maher, P.; Schubert, D. J. Neurochem. 2008, 105, 1336–1345.

Figure 1. Mass spectra of model and modified Aβ1-6 peptides: (A) mass spectrum of native Aβ1-6 peptide on ESI-positive mode; (B) mass spectrum of modified Aβ1-6 corresponding to a 45 Da decrease in mass compared with the native peptide on ESI-positive mode; (C) mass spectrum of modified Aβ1-6 corresponding to an 89 Da decrease in mass compared with the native peptide on ESI-positive mode.

injection volume was 30 µL. The column temperature was 40 °C. The mass spectrometer was operated with an electrospray source in positive ionization and single-ion monitoring (m/z 774, 729, and 685) modes for analytical state. The ESI source conditions were nebulizer gas rate of 0.18 L/min, CDL temperature of 230 °C, block temperature of 200 °C, probe voltage of +3.5 kV, interface temperature of 250 °C, and 1 s event time, respectively, and were obtained from a nitrogen source (N2 Supplier model 24S, Anest Iwata Co., Yokohama, Japan). In data analysis state, the acquired chromatogram was shown using TIC mode in fragment table. This LC-MS system was operated using LC-MS solution ver. 3.41-324. Preparation of Aβ Peptide Solutions. Aβ model peptides were dissolved in their original vial with water and acetonitrile (50/50, v/v) by sonication for 30 s to give 1 mM solutions. The solutions were stored at -20 °C until use. This water/acetonitrile solution was already applied to prepare the Aβ1-16 and Aβ1-40 peptides for the LC/MS experiment.16 In this result, this solution was useful for solubility, stability and LC separation of these peptides. Therefore, this solution was applied to shorter Aβ1-6 than other Aβ peptides. The degradation of Aβ1-6 in water/acetonitrile was not observed by MS response during the experimental period (6 months). Preparation of Solutions for Metal-Catalyzed Oxidation. For optimal MCO conditions, various concentrations of Cu(II) (CuSO4), Fe(II) (FeSO4), Zn(II) (ZnSO4), ascorbic acid, catecholamine (dopamine, epinephrine, or norepinephrine), and acetylcholine were prepared using pure water as the solvent. Metal-Catalyzed Oxidation. In the optimal condition, MCO reactions were performed at 37 °C with 0.1 mM Aβ1-6 peptide, 0.01 mM Cu(II), and 1.0 mM ascorbic acid in 50 mM Tris-HCl/Tris buffered to pH of 7.4 for 90 min. The MCO

reactions were initiated by the addition of ascorbic acid. For the investigated conditions, MCO reactions were performed at 37 °C with 0.1 mM Aβ model peptide, 0.01 mM Zn(II) or Fe(II), and 1.0 mM reducing reagents (dopamine, epinephrine, norepinephrine, acetylcholine, cholesterol, or estradiol) in 50 mM Tris-HCl/Tris buffered to pH of 7.4 for 90 min. The MCO reactions were initiated by adding the reducing reagents. Hydrogen Peroxide Oxidation. H2O2 reactions were performed at 37 °C with 0.1 mM Aβ model peptide and 1.0 mM H2O2 in 50 mM Tris-HCl/Tris buffered to pH of 7.4 for 90 min. This reaction was initiated by adding H2O2. Solid-Phase Extraction to Stop the Metal-Catalyzed or H2O2 Oxidation. Reactions were terminated by removing the substrates using solid-phase extraction (SPE). An OASIS-HLB (1 mL, 30 mg, Waters Co., Milford, MA) cartridge was used. Before extracting the reacted solutions, the SPE cartridge was conditioned by eluting 1.0 mL of methanol followed by 1.0 mL of 1% aqueous TFA. After the MCO reaction, the sample solution was diluted 2-fold with 1.0% aqueous TFA and eluted through an SPE cartridge. The cartridge was then washed with 1.0 mL of water. Methanol was added at a low flow rate to elute the peptides that were retained in the cartridges. The solutions were evaporated to dryness at 30 °C. The sample volumes were then adjusted to 1.0 mL of water/methanol (80/20, v/v) and measured by LC-MS. Screening Assay for Inhibitors of Metal-Catalyzed Oxidative Modification. To evaluate the MCO inhibitors, the optimal reaction was performed at 37 °C with 0.1 mM Aβ model peptide, 0.01 mM Cu(II) (CuSO4), test substances (0.01, 0.1, and 1.0 mM), and 1.0 mM ascorbic acid in 50 mM Tris-HCl/Tris buffered to pH of 7.4 for 90 min. The MCO reactions were initiated by the addition of ascorbic acid. The reaction was terminated by SPE and measured using LC-MS. In this Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

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Figure 2. LC-MS-SIM chromatograms of native and modified Aβ1-6 peptides under metal-catalyzed and H2O2 oxidation reactions: (A) MCO modification of model peptides (0.1 mM) in 0.01 mM Cu(II)/1.0 mM ascorbic acid in Tris-HCl buffer (pH 7.4); (B) H2O2 oxidation of model peptides (0.1 mM) in 1.0 mM H2O2 in Tris-HCl buffer (pH 7.4).

experiment, the test substances were BHT, curcumin, β-carotene, tocopherol, estradiol, and nicotine. Test substance solutions at concentrations of 0.01, 0.1, and 1.0 mM were obtained by adding DMSO to different test tubes. A control sample was prepared without test substances in DMSO and was used determine the background MCO inhibition level. Antioxidant activity for MCO was expressed as the inhibition value and was calculated using the following formula: A (control value) ) [(SIM response of m/z 685 + 729; modified model peptides in control sample)/ (SIM response of m/z 774; native model peptide in control sample)] B (test value) ) [(SIM response of m/z 685 + 729; modified model peptides in test sample)/ (SIM response of m/z 774; native model peptide in test sample)] antioxidant activity for MCO of peptide (%) ) [(A - B)/A] × 100 All analyses were performed three times, and their results are presented as the mean ± standard deviation. If the test substance had antioxidant activity toward MCO, these values would be significantly increased (up to 100%). On the other hand, if the substance did not have antioxidant activity, these values would be only slightly altered by the test substances. RESULTS AND DISCUSSION On the basis of a previous LC-MS assay of Aβ peptides, an initial experiment was performed to determine the retention time and MS ionizations of the native Aβ model peptide.16 The full scan mass spectrum was evaluated to determine the relative intensities of ions in a given mass range with positive ESI mode. The mass spectrum of the native Aβ1-6 showed a signal at m/z 774.1 and a doubly charged ion at m/z 387.9 (Figure 1A). The m/z 774.1 [M + H]+ on a single-ion monitoring (SIM) mode, the optimal LC mobile phase, column, and gradient conditions were used 1822

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to evaluate the Aβ1-6 model peptide and others after MCO, because then other peaks could be more clearly detected. After MCO of Aβ1-6, two novel oxidized peptides were observed with retention times of 13.0 and 15.5 min. These two oxidized peptides were relatively slow retention time to native peptide. This chromatographic phenomenon indicated the irreversible structural and polarity change of peptides. When subjected to MCO modification with Cu(II)/ascorbic acid, the singly and doubly charged Aβ1-6 were observed at m/z 729.2 and 364.8 and at m/z 685.3 and 343.3, corresponding to a decrease in mass of 45 and 89 Da compared with the Aβ model peptide (Figure 1, parts B and C). In contrast, the H2O2 reaction did not modify Aβ1-6 (Figure 2). Therefore, the modified peptides were characterized by a specific MCO reaction of Aβ1-6 that contained both His and N-terminal aspartate (Asp) residues. MS/MS evidence from MCO of Aβ1-16 peptides revealed that Cu(II) coordinates to the three His residues and suggests that an oxygen ligand comes from a carboxyl or hydroxyl side chain.10,19 In addition, the decarboxylation and deamination of N-terminal Asp to pyruvate (-45 Da) was detected by time-offlight MS following the MCO of the Aβ1-16 peptide.34 Peptides containing His residues underwent spontaneous N-terminal Asp oxidative decarboxylation in aqueous buffers with metal ions.35-37 Hawkins and Davies suggested that the alkoxyl radical pathway of peptide modification (-89 Da) is carried out with metal iondependent systems, where metal ion binding and other reactive species may have a role.38 On the basis of these reports, the MCOinduced modification of peptides containing both N-terminal Asp and His residues creates a characteristic change of the Aβ peptides. Aβ1-6 peptides were not modified by an H2O2 oxidative reaction (Figure 2). (34) Kowalik-Jankowska, T.; Ruta, M.; Wis´niewska, K.; Łankiewicz, L.; Dyba, M. J. Inorg. Biochem. 2004, 98, 940–950. (35) Kim, M. S.; Jeon, J. W.; Suh, J. J. Biol. Inorg. Chem. 2005, 10, 364–372. (36) Kim, M. G.; Kim, M. S.; Lee, S. D.; Suh, J. J. Biol. Inorg. Chem. 2006, 11, 867–875. (37) Levine, J.; Etter, J.; Apostol, I. J. Biol. Chem. 1999, 274, 4848–4857. (38) Hawkins, C. L.; Davies, M. J. Biochim. Biophys. Acta 2001, 1504, 196– 219.

Figure 3. LC-MS-SIM chromatograms of Aβ1-6 and modified peptides under various conditions with metal ions and reducing agents: (A) MCO with 0.01 mM Cu(II)/1.0 mM ascorbic acid in Tris-HCl buffer (pH 7.4); (B) MCO with 0.01 mM Zn(II)/1.0 mM ascorbic acid in Tris-HCl buffer (pH 7.4); (C) MCO with 0.01 mM Fe(II)/1.0 mM ascorbic acid in Tris-HCl buffer (pH 7.4); (D) MCO with 0.01 mM Cu(II)/1.0 mM dopamine in Tris-HCl buffer (pH 7.4); (E) MCO with 0.01 mM Cu(II)/1.0 mM epinephrine in Tris-HCl buffer (pH 7.4); (F) MCO with 0.01 mM Cu(II)/1.0 mM norepinephrine in Tris-HCl buffer (pH 7.4); (G) MCO with 0.01 mM Cu(II)/1.0 mM acetylcholine in Tris-HCl buffer (pH 7.4); (H) MCO with 0.01 mM Cu(II)/1.0 mM cholesterol in Tris-HCl buffer (pH 7.4); (I) MCO with 0.01 mM Cu(II)/1.0 mM estradiol in Tris-HCl buffer (pH 7.4).

The terminal MCO reaction with Cu(II)/ascorbic acid has been applied to acetic acid.16 Two modified Aβ1-6 peptides were confirmed by LC-MS-SIM, but these samples were not sufficient for terminal reactions after storage in acetic acid. Moreover, the use of various solutions such as Tris-HCl/Tris buffer, acid solutions, and DMSO should have adverse effect (signal suppression) in the mass spectral signal. Thus, we used SPE to eliminate the metal ions and other reagents instead of adding acetic acid for stable stopping procedure and negating an effect of MS ionization. In studies of SPE’s optimization, the Bond Elut-C18 (100 mg, 1 mL, Varian) and OASIS-HLB (30 mg, 1 mL, Waters) were investigated with water, acetic acid, and TFA solutions. For Bond Elut-C18, the recovery of Aβ1-6 was less than 50%. On the other hand, good recovery (90-98.5%) using OASIS HLB was achieved using TFA solutions. In addition, matrix effects of these peptides could be disappeared with this SPE procedure. An LC-MS-SIM chromatogram of MCO-reacted Aβ1-6 peptides is shown in Figure 2. LC-MS-SIM with SPE was a useful and simple technique for detecting the amounts of Aβ1-6 and modified peptides in an MCO reaction. Then, we studied the effective metal substrate reactivity for MCO modification of Aβ1-6. Bush reported that the metallochemistry of Zn, Fe, and Cu to mediate aggregation and

modification of Aβ peptides was an important factor of the AD hypothesis.2 Thus, we investigated MCO of Aβ1-6 peptide with the advanced metal agents Zn(II), Fe(II), and Cu(II) using LC-MS-SIM analysis. In this result, the modification of the Aβ1-6 peptide could be not observed in MCO reactions with Zn(II) or Fe(II)/ascorbic acid (Figure 3A-C). These results indicated that this MCO of Aβ1-6 occurred with specific Cu(II) and reducing agents without Zn(II) and Fe(II). We also tested the efficacy of reducing agents on MCOinduced modification of Aβ1-6. Opazo et al. reported that the Aβ peptide was not toxic in cell cultures in the absence of Cu(II) ions and that the presence of dopamine markedly exaggerated the neurotoxicity of Aβ1-42.39 Thus, we evaluated the MCO of the model peptide with advanced catecholaminergic agents along with Cu(II) using LC-MS-SIM analysis. The chromatograms of the modified peptides with various reducing agents with Cu(II) are shown in Figure 3. Little modification of the Aβ1-6 peptide was observed in the MCO with Cu(II)/dopamine and norepinephrine (Figure 3, parts D and F). MCO with Cu(II)/epinephrine could modify Aβ1-6 (Figure 3E). On the other hand, MCO with Cu(II)/other reducing agents did not modify Aβ1-6 (Figure 3G-I). Unlike norepinephrine, epinephrine is known to be rapidly oxidized.40 This report is in keeping with the concept that epinephrine Analytical Chemistry, Vol. 81, No. 5, March 1, 2009

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Figure 4. Investigation of optimal concentrations of Cu(II) ion and ascorbic acid, and MCO reaction time: (A) effect of ascorbic acid concentration (0.1, 0.5, and 1.0 mM) on MCO with 0.01 mM Cu(II) for 90 min; (B) effect of Cu(II) concentration (0.001, 0.005, 0.01 and 0.05 mM) on MCO with 1.0 mM ascorbic acid for 90 min; (C) effect of reaction time (0-180 min) on MCO with 1.0 mM ascorbic acid/ 0.01 mM Cu(II).

plays a key role in reducing Cu(II) to Cu(I) in order to initiate a one-electron reduction of molecular oxygen. On the basis of the chromatographic responses of the modified peptides, the Cu(II)/ascorbic acid system was the most effective reducing agent of MCO-induced modification of Aβ1-6. Finally, we investigated the dose-response and reaction time of MCO with Cu(II)/ascorbic acid. Ascorbic acid concentrations were changed while maintaining the Cu(II) concentration at 0.01 mM for 90 min (Figure 4A). The ascorbic acid had little effect until reaching a concentration of 0.1 mM. Next, Cu(II) concentrations were changed while maintaining the ascorbic acid concentration at 1.0 mM for 90 min (Figure 4B). Aβ1-6 was modified at a Cu(II) concentration of 0.01 mM. Therefore, the optimal conditions for MCO modification of the Aβ1-6 (0.1 mM) were 0.01 mM Cu(II)/ 1.0 mM ascorbic acid in Tris-HCl buffer (pH 7.4). Under the optimal MCO condition, the reaction was fully saturated at 90 min (Figure 4C). However, if 1.0 mM ascorbic acid was added to fully saturated reaction in the tube, the MCO could (39) Opazo, C.; Huang, X.; Cherny, R. A.; Moir, R. D.; Roher, A. E.; White, A. R.; Cappai, R.; Masters, C. L.; Tanzi, R. E.; Inestrosa, N. C.; Bush, A. I. J. Biol. Chem. 2002, 277, 40302–40308. (40) Costa, V. M.; Silva, R.; Ferreira, L. M.; Branco, P. S.; Carvalho, F.; Bastos, M. L.; Carvalho, R. A.; Carvalho, M.; Remia˜o, F. Chem. Res. Toxicol. 2007, 20, 1183–1191.

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occur after 90 min. Thus, the MCO occurs the completely consumed 1.0 mM ascorbic acid after 90 min. The results of these experiments indicated that the LC-MS assay with SPE can be utilized to monitor MCO-induced modification of model peptides with Cu(II)/ascorbic acid in Tris-HCl (pH 7.4) for 90 min. Using LC-MS-SIM detection of modified peptides, we developed a screening assay to identify antioxidants that inhibit MCO modification of a model peptide, because the inevitable progression from the H2O2 reaction to MCO-induced oxidative damage to peptides and/or proteins in a state of oxidative stress must be further examined. A recent paper suggested that H2O2 also affects numerous intracellular normal signaling pathways.41 Thus, the ROS redox reaction might be implicated in the cellular signaling that regulates normal processes and the progression of aging-related diseases, including AD, and brain dysfunction.42 Moreover, Opazo et al. suggested that microregional catalytic ROS production the depletion of reducing agents may mediate the neurotoxicity of Aβ in AD, and inhibitors of this activity may be of therapeutic value.39 Therefore, our identification of the MCO-induced modification of Aβ1-6 in simple physiologic condition would be useful for inhibitor’s assay of specific antioxidants for the involvement of the ROS reaction with MCO. To evaluate the substances that inhibit MCO, MCO was induced with the Aβ1-6, Cu(II), test substances (0.01, 0.1, and 1.0 mM), and ascorbic acid in Tris-HCl buffered to pH 7.4 for 90 min. The reaction was then terminated by SPE and measured by LC-MS. The antioxidant activity against MCO modification of peptides was calculated using LC-MS-SIM response ratios of modified peptides (m/z 729 + 685) per native (m/z 774) model peptide in control and test reactions (Figure 5). Antioxidant test substances were selected based on their reported antiamyloidogenic effects.26 Test substances such as BHT, carotene, tocopherol, estradiol, and nicotine did not inhibit MCO-induced Aβ modification with Cu(II)/ascorbic acid. On the other hand, curcumin at concentrations of 0.01 (17.8%), 0.1 (63.6%), and 1.0 mM (79.4%) inhibited MCO of model peptides (Figure 5). Thus, on the basis of the findings obtained using this novel assay, there are obvious major differences between curcumin and other known antioxidants. Curcumin has recently attracted great interest because of its important pharmacologic activities, particularly its anti-inflammatory, anticarcinogenic, and anti-AD activities.43 On the basis of our data (Figure 5), curcumin has potential therapeutic roles as inhibitors of oxidative stress in the brain. Pharmacokinetic studies suggest that curcumin moves from the bloodstream to the brain, where it can interact with Aβ plaques.44,45 Therefore, our assay to screen inhibitors of MCO may be useful for determining other novel and interesting effects of curcumin related to preventing metalloenzyme-like activity. (41) Lambeth, J. D. Nat. Rev. Immunol. 2004, 4, 181–189. (42) Rhee, S. G. Science 2006, 312, 1882–1883. (43) Goel, A.; Kunnumakkara, A. B.; Aggarwal, B. B. Biochem. Pharmacol. 2008, 75, 787–809. (44) Pan, M. H.; Huang, T. M.; Lin, J. K. Drug. Metab. Dispos. 1999, 27, 486– 494. (45) Ryu, E. K.; Choe, Y. S.; Lee, K. H.; Choi, Y.; Kim, B. T. J. Med. Chem. 2006, 49, 6111–6119.

Figure 5. Antioxidant activity against MCO modification of Aβ1-6 peptides using LC-MS-SIM response ratios of modified peptides (m/z 729 + 685) per native (m/z 774) peptide in control and test (0.01, 0.1, and 1.0 mM) reactions.

assays using model peptides are able to directly detect Aβ fibril formation and aggregation.27,47-49 No analytical method to date, however, targets the specific pathogenesis induced by the ROS produced by metal-binding peptides. In the present study, we focused on the progression of oxidative damage of N-terminal Aβ1-6 peptide in a state of oxidative stress produced by MCO and developed an assay to screen inhibitors of this reaction using LC-MS (Figure 6). This novel and simple screening assay of potential MCO inhibitors allowed for the discovery of the interesting effects of curcumin. Thus, the use of this newly developed assay may allow us to determine singular and/or combined antioxidants such as flavonoides and catechines that are effective against MCO of peptides and/or proteins related to degenerative diseases. Moreover, it is needed that the cause-andeffect sequence of Aβ aggregated events with MCO would be investigated using this simple phenomenon. Figure 6. Illustration of this assay for evaluating inhibitors of MCO in the model peptide.

CONCLUSIONS To screen for novel therapeutic strategies for preventing the pathologic conditions of AD, various model peptide systems have been utilized to evaluate the modification, formation, and aggregation of the Aβ peptide.46 Almost all previously developed analytical (46) Esteras-Chopo, A.; Pastor, M. T.; Lo´pez de la Paz, M. Methods Mol. Biol. 2006, 340, 253–276.

Received for review December 31, 2008.

October

12,

2008.

Accepted

AC802162N (47) Inbar, P.; Bautista, M. R.; Takayama, S. A.; Yang, J. Anal. Chem. 2008, 80, 3502–3506. (48) Ryu, J.; Joung, H.-A.; Kim, M.-G.; Park, C. B. Anal. Chem. 2008, 80, 2400– 2407. (49) Kato, M.; Kinoshita, H.; Enokita, M.; Hori, Y.; Hashimoto, T.; Iwatsubo, T.; Toyo’oka, T. Anal. Chem. 2007, 79, 4887–4891.

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