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• W2013396018 abstract "The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b5 (b5) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an 18O-labeling method on the basis that cross-linked peptides incorporate twice as many 18O atoms as non-cross-linked peptides during proteolysis conducted in 18O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys428(CYP2E1)-Asp53(b5) and Lys434(CYP2E1)-Glu56(b5), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b5 interaction, and the stimulatory effect of b5, was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b5 complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations. The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b5 (b5) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an 18O-labeling method on the basis that cross-linked peptides incorporate twice as many 18O atoms as non-cross-linked peptides during proteolysis conducted in 18O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys428(CYP2E1)-Asp53(b5) and Lys434(CYP2E1)-Glu56(b5), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b5 interaction, and the stimulatory effect of b5, was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b5 complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations. Cytochrome P450s (P450s) 2The abbreviations used are: P450s, cytochrome P450s; CYP2E1, E. coli-expressed recombinant human cytochrome P450 2E1; human b5, E. coli-expressed recombinant human cytochrome b5; rat b5, E. coli-expressed recombinant rat cytochrome b5; P450 reductase, NADPH-dependent cytochrome P450 oxidoreductase; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; ESI-QTOF MS, electrospray ionization quadrupole time-of-flight mass spectrometry; IT-FT-ICR MS, hybrid ion trap-Fourier transform ion cyclotron resonance mass spectrometry; CID, collision-induced dissociation; MS/MS, tandem mass spectrometry; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DLPS, 1,2-dilauroyl-sn-glycero-3-phospho-l-serine; APAP, acetaminophen; PNP, p-nitrophenol; GS-APAP, 3′-glutathione-S-yl-APAP. are a superfamily of b-type hemoproteins responsible for the metabolism of a wide variety of exogenous compounds such as drugs and carcinogens, and endogenous compounds such as prostaglandins and steroids (1.Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2663) Google Scholar). P450 reactions require input of two electrons (supplemental Fig. S1) (1.Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2663) Google Scholar, 2.Yun C.H. Miller G.P. Guengerich F.P. Biochemistry. 2001; 40: 4521-4530Crossref PubMed Scopus (37) Google Scholar). The efficiency of electron transfer is one of the key determinants of the reaction kinetics. In microsomal systems, NADPH is the ultimate source of the two electrons, and NADPH-dependent cytochrome P450 oxidoreductase (P450 reductase) together with cytochrome b5 (b5) facilitates the electron transfer. Knowledge of the interactions between P450s and their redox partners is fundamental to a complete understanding of the mechanisms of P450 reactions. The interactions between P450s and b5 have drawn much attention because of variable effects b5 has on different P450 isoforms and P450 reactions. It has been shown that b5 may stimulate, inhibit or have no effects on P450-catalyzed reactions depending on the particular isoform of P450 and the substrate of the reaction (3.Schenkman J.B. Jansson I. Pharmacol. Ther. 2003; 97: 139-152Crossref PubMed Scopus (382) Google Scholar, 4.Schenkman J.B. Jansson I. Drug Metab. Rev. 1999; 31: 351-364Crossref PubMed Scopus (65) Google Scholar, 5.Porter T.D. J. Biochem. Mol. Toxicol. 2002; 16: 311-316Crossref PubMed Scopus (154) Google Scholar). However, there is no consensus on whether b5 transfers electrons to P450, or causes an allosteric effect on P450, or whether both mechanisms are simultaneously operative (6.Yamazaki H. Johnson W.W. Ueng Y.F. Shimada T. Guengerich F.P. J. Biol. Chem. 1996; 271: 27438-27444Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 7.Perret A. Pompon D. Biochemistry. 1998; 37: 11412-11424Crossref PubMed Scopus (81) Google Scholar, 8.Yamazaki H. Shimada T. Martin M.V. Guengerich F.P. J. Biol. Chem. 2001; 276: 30885-30891Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 9.Gilep A.A. Guryev O.L. Usanov S.A. Estabrook R.W. J. Inorg. Biochem. 2001; 87: 237-244Crossref PubMed Scopus (22) Google Scholar, 10.Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (81) Google Scholar). In addition, the mechanism of substrate- and P450 isoform dependency is unknown (5.Porter T.D. J. Biochem. Mol. Toxicol. 2002; 16: 311-316Crossref PubMed Scopus (154) Google Scholar, 11.Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expr. Purif. 2002; 24: 329-337Crossref PubMed Scopus (215) Google Scholar). CYP2E1 is a P450 isoform whose reactions are highly stimulated in the presence of b5. For example, we previously found that b5 stimulates CYP2E1-catalyzed oxidation of acetaminophen to its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by 25-fold (12.Chen W. Koenigs L.L. Thompson S.J. Peter R.M. Rettie A.E. Trager W.F. Nelson S.D. Chem. Res. Toxicol. 1998; 11: 295-301Crossref PubMed Scopus (206) Google Scholar). For other CYP2E1-catalyzed reactions, such as aniline p-hydroxylation and 7-ethoxycoumarin O-deethylation, b5 stimulates the reactions by 270-fold and 67-fold, respectively (5.Porter T.D. J. Biochem. Mol. Toxicol. 2002; 16: 311-316Crossref PubMed Scopus (154) Google Scholar, 11.Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expr. Purif. 2002; 24: 329-337Crossref PubMed Scopus (215) Google Scholar). In contrast to its stimulating effects on CYP3A4, CYP3A5, CYP2C19, CYP2B6, and CYP2C8, apo-b5 is unable to replace holo-b5 in stimulating CYP2E1-catalyzed reactions (11.Yamazaki H. Nakamura M. Komatsu T. Ohyama K. Hatanaka N. Asahi S. Shimada N. Guengerich F.P. Shimada T. Nakajima M. Yokoi T. Protein Expr. Purif. 2002; 24: 329-337Crossref PubMed Scopus (215) Google Scholar, 12.Chen W. Koenigs L.L. Thompson S.J. Peter R.M. Rettie A.E. Trager W.F. Nelson S.D. Chem. Res. Toxicol. 1998; 11: 295-301Crossref PubMed Scopus (206) Google Scholar). The requirement for the heme group increases the probability that b5 stimulates CYP2E1-catalyzed reactions by facilitating electron transfer rather than by only causing a positive allosteric effect. Because intermolecular complex formation immediately precedes electron transfer(13.Rodgers K.K. Sligar S.G. J. Mol. Biol. 1991; 221: 1453-1460Crossref PubMed Scopus (82) Google Scholar), the identification of the protein interacting regions and orientations in the CYP2E1-b5 complex is an important goal in understanding the effects of b5 on CYP2E1-catalyzed reactions. It has been shown, by site-directed mutagenesis and chemical modification, that several cationic residues on the proximal face of P450, and several anionic residues on b5 surrounding the solvent-exposed heme edge, are important for the functional interaction between the two proteins (4.Schenkman J.B. Jansson I. Drug Metab. Rev. 1999; 31: 351-364Crossref PubMed Scopus (65) Google Scholar, 14.Bridges A. Gruenke L. Chang Y.T. Vakser I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 15.Hlavica P. Schulze J. Lewis D.F. J. Inorg. Biochem. 2003; 96: 279-297Crossref PubMed Scopus (68) Google Scholar). However, the protein interacting surfaces have not been fully characterized, and the protein interacting orientations have never been determined. One complex model of b5 and CYP101, a microbial P450, was proposed by Sligar and co-workers (16.Stayton P.S. Poulos T.L. Sligar S.G. Biochemistry. 1989; 28: 8201-8205Crossref PubMed Scopus (131) Google Scholar) based on visual optimization of the intermolecular electrostatic interactions and minimization of the distance between the redox centers of the two proteins. However, the protein interacting orientations in this model have not been substantiated by experiments. In this study, a complex model of b5 and CYP2E1, a microsomal P450, is proposed based on two chemical crosslinks characterized using mass spectrometry. Because electrostatic interactions are considered the major driving forces for the P450-b5 interaction (4.Schenkman J.B. Jansson I. Drug Metab. Rev. 1999; 31: 351-364Crossref PubMed Scopus (65) Google Scholar, 14.Bridges A. Gruenke L. Chang Y.T. Vakser I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 15.Hlavica P. Schulze J. Lewis D.F. J. Inorg. Biochem. 2003; 96: 279-297Crossref PubMed Scopus (68) Google Scholar), a water-soluble cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was chosen to covalently link CYP2E1 with b5. EDC generates “zero-length” cross-links (amide bonds) between basic (Lys) and acidic (Asp or Glu) residues that come into very close proximity (supplemental Fig. S2) (17.Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar). The short length of EDC cross-links generally leads to specific intra- and intermolecular linkages without sampling multiple protein orientations (17.Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (726) Google Scholar). EDC was previously used to form a cross-link between CYP2B4 and b5 (18.Tamburini P.P. Schenkman J.B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 11-15Crossref PubMed Scopus (53) Google Scholar). However, this complex was not structurally characterized. To identify the cross-links in the CYP2E1-b5 complex, the complex was digested and the generated peptides were isotopically labeled. It has been noted that during trypsin-catalyzed proteolysis, two oxygen atoms from solvent are incorporated into the α-carboxyl group of a peptide C-terminally ending with a lysine or an arginine residue (19.Schnolzer M. Jedrzejewski P. Lehmann W.D. Electrophoresis. 1996; 17: 945-953Crossref PubMed Google Scholar). When the proteolysis is conducted in fully enriched 18O-water, the generated peptides are labeled with two 18O atoms at their C termini (Fig. 1) (19.Schnolzer M. Jedrzejewski P. Lehmann W.D. Electrophoresis. 1996; 17: 945-953Crossref PubMed Google Scholar, 20.Yao X. Freas A. Ramirez J. Demirev P.A. Fenselau C. Anal. Chem. 2001; 73: 2836-2842Crossref PubMed Scopus (779) Google Scholar). Recently, 18O-labeling has been used to identify crosslinked peptides in a digest mixture (21.Back J.W. Notenboom V. de Koning L.J. Muijsers A.O. Sixma T.K. de Koster C.G. de Jong L. Anal. Chem. 2002; 74: 4417-4422Crossref PubMed Scopus (127) Google Scholar, 22.Collins C.J. Schilling B. Young M. Dollinger G. Guy R.K. Bioorg. Med. Chem. Lett. 2003; 13: 4023-4026Crossref PubMed Scopus (49) Google Scholar). Through the comparison of peptide ions generated from two digestions, one conducted in 16O-water and the other in 18O-water, cross-linked peptides can be distinguished from non-cross-linked peptides by virtue of incorporating more than two 18O atoms during proteolysis. The structures of the selected cross-linked peptide candidates are subsequently characterized by tandem mass spectrometric (MS/MS) analysis. Here we describe the characterization of two cross-links in the CYP2E1-b5 complex using 18 O-la-importance of the identified ion pairs in the interacting proteins was confirmed by site-directed mutagenesis. Finally, a model that sheds light on the CYP2E1-b5 interaction was constructed. Materials—Escherichia coli strain DH5α containing the expression vector pCWhum3A4(His)6 and strain BL21(DE3) containing the plasmid (His)4HMwRat-b5 were provided by Dr. Ronald W. Estabrook (10.Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (81) Google Scholar, 23.Holmans P.L. Shet M.S. Martin-Wixtrom C.A. Fisher C.W. Estabrook R.W. Arch. Biochem. Biophys. 1994; 312: 554-565Crossref PubMed Scopus (101) Google Scholar). E. coli strain DH5α containing the expression vector pCWhum2E1 was from Dr. Elizabeth M. J. Gillam (24.Gillam E.M. Guo Z. Guengerich F.P. Arch. Biochem. Biophys. 1994; 312: 59-66Crossref PubMed Scopus (150) Google Scholar). Restriction enzymes and other DNA-modifying enzymes were from New England BioLabs (Beverly, MA). Platinum Pfx DNA polymerase, T4 DNA ligase, E. coli DH5αF'IQ Max Efficiency Competent cells, E. coli expressed histidine-tagged recombinant human b5 were purchased from Invitrogen (Carlsbad, CA). Bactotryptone, bactopeptone, and bactoyeast extract were obtained from BD Biosciences Clontech (Palo Alto, CA). Emulgen 911 was from Kao Chemicals (Tokyo, Japan). Ni-NTA Superflow was from Qiagen (Valencia, CA). The cross-linking reagent EDC was purchased from Pierce. Sequencing grade modified trypsin was from Roche Applied Science (Indianapolis, IN). 18O-labeled water (99 atom % 18O) was purchased from Isotec (Miamisburg, OH). IPTG, δ-ALA, thiamine, imidazole hydrochloride, protease inhibitor mixture, sodium cholate, Coomassie Brilliant Blue R, Copper II chloride dehydrate, DTT, and iodoacetamide were purchased from Sigma-Aldrich. HPLC solvents were of the highest grade commercially available and were used as received. All other reagents were analytical grade. Cloning of Histidine-tagged Recombinant Human CYP2E1—The vector construct pCWhum3A4(His)6 (10.Guryev O.L. Gilep A.A. Usanov S.A. Estabrook R.W. Biochemistry. 2001; 40: 5018-5031Crossref PubMed Scopus (81) Google Scholar) was the source of the pCWori+ expression vector for cloning the histidine-tagged CYP2E1 expression vector construct. The cDNA of human CYP2E1 (24.Gillam E.M. Guo Z. Guengerich F.P. Arch. Biochem. Biophys. 1994; 312: 59-66Crossref PubMed Scopus (150) Google Scholar) was used as the template for PCR. Amplifications were performed using Platinum Pfx DNA polymerase. Reactions were assembled and heated to 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 56 °C for 45 s, and extension at 72 °C for 1 min. Cycling was followed by a final extension at 72 °C for 5 min. DNA from the reactions was purified using a Qiagen PCR purification kit and digested with NdeI and SalI. The digests were then electrophoresed on a 1.1% agarose gel and the CYP2E1 amplicons of expected size were gel-purified using a Qiagen Geneclean kit. The pCWhum3A4(His)6 plasmid was digested with NdeI and SalI to enable gel-purification of the linearized plasmid from which the CYP3A4 insert had been removed. The vector was treated with calf intestinal phosphatase and the CYP2E1 fragment was ligated to the vector to generate the histidine-tagged CYP2E1 expression vector construct. Ligation reactions were used to transform DH5α cells. Positive clones containing the desired inserts were verified initially by colony PCR and restriction analysis, and finally by DNA sequencing using the dideoxy chain termination method. Site-directed Mutagenesis of CYP2E1—The cloned plasmid pCWhum2E1(His)6 was used as the template for amplification reactions with the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Oligonucleotide primers used in the generation of K428A and K434A single mutants plasmids were as follows (mismatches indicated by the underlined bases): K428A forward, 5′-GTGACTATTTCGCGCCATTTTCCAC-3′; K428A reverse, 5′-GTGGAAAATGGCGCGAAATAGTCAC-3′. K434A forward, 5′-AAGCCATTTTCCACAGGAGCACGAGTGTGTGCTGGAGAA-3′; K434A reverse, 5′-TTCTCCAGCACACACTCGTGCTCCTGTGGAAAATGGCTT-3′. A double mutant of pCWhum2E1(His)6 containing both the K428A and the K434A replacements was constructed using the K434A single mutant plasmid and the K428A forward and reverse primers. DpnI-digested DNA was transformed into XL1-Blue cells, and DNA from several of the resulting colonies was isolated. The cDNA sequence was analyzed for the presence of the desired mutations and the absence of extraneous mutations (University of Washington Sequencing Facility). Protein Expression and Purification—A single isolated colony of histidine-tagged CYP2E1 was used to inoculate 10 ml of LB-ampicillin media, which was cultivated with shaking at 37 °C overnight and then diluted 1:100 in Terrific Broth containing 100 mg of ampicillin liter−1, 1.0 mm thiamine, and trace elements (25.Bauer S. Shiloach J. Biotechnol. Bioeng. 1974; 16: 933-941Crossref PubMed Scopus (101) Google Scholar). The cultures were shaken (180 rpm) at 37 °C until the A600 reached 0.4, after which isopropyl-1-thio-β-d-galactopyranoside (1.0 mm) and δ-ALA (0.5 mm) were added, and the cultures were shaken (160 rpm) at 28 °C for 36 h. Cells were harvested by centrifugation at 5000 × g (4 °C, 15 min), resuspended in storage buffer (50 mm KPi, pH 7.4, 20% glycerol, and 0.5 mm EDTA), pooled into 50-ml Falcon tubes, and recentrifuged at 4000 × g (4 °C, 30 min). The supernatant was discarded, and the cell pellets were resuspended in resuspension buffer (100 mm Tris-HCl, pH 7.4, 20% glycerol, with the addition of 1 ml of protease inhibitor mixture per liter of the initial culture volume). After the addition of lysozyme (5 mg/liter), the culture was stirred at 4 °C for 1 h. Cells were homogenized and spun at 150,000 × g. The pellets were resuspended in the resuspension buffer by homogenization and stirred at 4 °C for 1 h after the addition of 1% Emulgen 911. After centrifugation at 150,000 × g for 25 min, imidazole was added to the red/orange supernatant to a final concentration of 20 mm. The supernatant was applied to a Ni-NTA agarose column that had been pre-equilibrated with 15 column volumes of equilibrium buffer (50 mm KPi, pH 7.4, 20% glycerol, 0.5 m KCl, 0.05% sodium cholate, 50 μm α-NF, and protease inhibitors). The column was washed with 20 column volumes of washing buffer A (50 mm KPi, pH 7.4, 20% glycerol, 40 mm imidazole, 0.05% sodium cholate, 0.02 mm DTT, and protease inhibitors). CYP2E1 enzyme was eluted with elution buffer A (50 mm KPi, pH 7.4, 20% glycerol, 350 mm imidazole, and 0.02% sodium cholate). The eluted fractions were dialyzed against dialysis buffer A (100 mm KPi, pH 7.4, 20% glycerol, 0.5 mm EDTA, and 0.1 mm DTT). The dialysate was applied slowly (20 ml/hr) to a hydroxyapatite column (1.5 × 3 cm) and washed with 5 column volumes of washing buffer B (50 mm KPi, pH 7.4, 20% glycerol, 0.5 mm EDTA, and 0.1 mm DTT). The CYP2E1 enzyme was eluted with elution buffer B (300 mm KPi, pH 7.4, 20% glycerol) and dialyzed against dialysis buffer B (50 mm KPi, pH 7.4, 20% glycerol). Rat b5 expression plasmid was kindly provided by Dr. Ronald W. Estabrook and expressed in BL21-DE3 cells using previously described conditions (23.Holmans P.L. Shet M.S. Martin-Wixtrom C.A. Fisher C.W. Estabrook R.W. Arch. Biochem. Biophys. 1994; 312: 554-565Crossref PubMed Scopus (101) Google Scholar). Expression and purification of P450 reductase was accomplished as previously described (26.Shen A.L. Christensen M.J. Kasper C.B. J. Biol. Chem. 1991; 266: 19976-19980Abstract Full Text PDF PubMed Google Scholar). Cross-linking Reactions—All enzymes used for cross-linking reactions were dialyzed against dialysis buffer B. CYP2E1, b5 (human b5 or rat b5) and DLPC were reconstituted with molar ratio of 1:1:500. The solution was gently stirred for 10 min and held at room temperature for 2 h. EDC was added to 8 mm final concentration from a 100 mm stock. The reaction was allowed to proceed at room temperature for 2 h. Proteolytic Digestions—For in-gel proteolysis, the cross-linking reaction was quenched by the addition of an equal volume of 2 × SDS loading buffer containing 200 mm DTT. SDS-PAGE was performed, and the protein gels were stained by either Coomassie Blue or copper II chloride dihydrate. Copper staining was carried out by soaking the gel in 300 mm CuCl2. When the desired degree of opacity was reached, the staining solution was removed, and the gel was kept in water. The band of interest was excised and washed three times with destaining buffer (25 mm Tris-HCl, 192 mm glycine, pH 8.3). After destaining, the gel piece was washed with 500 μl of 100 mm ammonium bicarbonate buffer (pH 8.5) for 10 min, dehydrated in 500 μl of acetonitrile at room temperature for 15 min, and dried in a Speed-Vac for 20 min. Subsequently, the gel was rehydrated with 300 μl of 100 mm ammonium bicarbonate buffer containing 10 mm DTT, and incubated at 56 °C for 50 min to reduce the disulfides. Then the gel piece was washed with 500 μl of 100 mm ammonium bicarbonate buffer (pH 8.5) for 10 min, dehydrated in 500 μl of acetonitrile at room temperature for 15 min, and dried in the SpeedVac for 20 min. To alkylate cysteine residues, the gel piece was rehydrated with 200 μl of 100 mm ammonium bicarbonate buffer containing 60 mm iodoacetamide and incubated in the dark at room temperature for 50 min. The gel piece was cut into two halves and both pieces were then washed with 500 μl of 100 mm ammonium bicarbonate buffer (pH 8.5) for 10 min, dehydrated in 500 μl of acetonitrile at room temperature for 15 min and dried in the SpeedVac. The dried gel pieces were rehydrated with two digestion solutions (50 mm ammonium bicarbonate, pH 8.5, with sequencing grade trypsin at an enzyme:substrate ratio of 1:25 (w/w), prepared with 16O- and 18O-water respectively. The digestion was allowed to proceed at 37 °C for 24 h, and the reaction was quenched with 0.1% trifluoroacetic acid. For post-proteolysis (a proteolysis step conducted in 16O-water followed by a post-digest labeling step carried out in 18O-water), subsequent to trypsin digestion in 16O-water, the peptide mixture was dried completely in the SpeedVac. Digestion solution prepared with 18O-water was then added, and the oxygen exchange reaction was allowed to proceed at 37 ° for 24 h. The reaction was quenched with 0.1% trifluoroacetic acid. For in-solution proteolysis, the cross-linking reaction was quenched by the removal of EDC through dialysis against dialysis buffer B. Glycerol was removed by a second dialysis against dialysis buffer C (50 mm KPi, pH 7.4). The sample was dried completely in the SpeedVac and resuspended in 6 m urea, 100 mm Tris-Base, pH 8.0, to yield a protein concentration ∼2 mg/ml. 50 μl of the protein sample was transferred to another microcentrifuge tube and reduced by adding 10 μl of 100 mm Tris-Base, pH 8.0, containing 100 mm DTT. The reaction was carried out at room temperature for 1 h. Subsequent alkylation reactions were initiated by adding 30 μl of 100 mm Tris-Base, pH 8.0, containing 500 mm iodoacetamide. The reactions were allowed to proceed in the dark at room temperature for 1 h. Samples were subsequently diluted with 50 mm ammonium bicarbonate and centrifuged using an Ultrafree ®-4 Centrifugal Filter Unit (Millipore, Billerica, MA). The dilution and centrifugation steps were repeated three times. Samples were then split into two equal aliquots, which were dried in the SpeedVac. The two dried peptide samples were reconstituted in two digestion solutions (prepared with 16O- and 18O-water respectively). The digestion was allowed to proceed at 37 °C for 24 h, and the reaction was quenched with 0.1% trifluoroacetic acid. ESI-QTOF MS Analysis—Protein mass spectra were recorded on an API-US quadrupole/time-of-flight (QTOF) mass spectrometer (Micromass, Manchester, UK). Protein samples were injected on a 300 μm i.d. × 5 cm perfusion column, packed with 20 μm POROS R2 particles (PerSeptive Biosystems, Framingham, MA), operated at a flow rate of 20μl/min and interfaced on-line with the QTOF mass spectrometer. Instrument parameters were as follows: source temperature, 100 °C; N2 drying gas, 50 liters/hr; electrospray voltage, 3.8 kV; and cone voltage, 60 V. Data acquisition was carried out from m/z 800-2400 using a 2.4 s scanning time. The gradient elution profile was set as follows: 5% solvent B for 2 min and 5-90% solvent B over the next 5 min. (solvent A, 5% acetonitrile, 0.1% trifluoroacetic acid; solvent B, 95% acetonitrile, 0.1% trifluoroacetic acid). Peptide digests were analyzed using the QTOF mass spectrometer equipped with a CapLC system (Waters, Milford, MA). The stream select module was configured witha5mm × 300 μm i.d. trap column packed with 5-μmC4 particles (LC Packings, San Francisco, CA) connected by a ZU1XC metallic union (Valco, Houston, TX) to a 20 cm × 75 μm i.d. nanoscale analytical column packed in-house with 5-μm Jupiter C18 particles (Phenomenex, Torrance, CA) using the method described by Kennedy and Jorgenson (27.Kennedy R.T. Jorgenson J.W. Anal. Chem. 1989; 61: 436-441Crossref PubMed Scopus (105) Google Scholar). Peptide samples were injected onto the trap column at 10 μl/min, cleaned-up and back-flushed to the analytical column at 0.5 μl/min using gradient elution. Binary gradients of 5-60% solvent B were generated over 30 min, followed by 60% B for 5 min and 60-90% B for 5 min (solvent A, 3.3% acetonitrile, 1.7% 2-propyl alcohol, and 0.1% trifluoroacetic acid; solvent B, 63.3% acetonitrile, 31.7% 2-propyl alcohol, and 0.1% trifluoroacetic acid). QTOF parameters were set as follows: electrospray potential, 3.6 kV; cone voltage, 35 V; and source temperature, 100 °C. The instrument was operated at a mass resolving power of 6,000. For MS/MS, the scan time was set to 2 s, the precursor isolation width set to 4 Da, and the collision energy set to 25-45 eV according to the m/z of the precursor and the charge state. IT-FT-ICR MS Analysis—Peptide digests were analyzed by electrospray ionization in the positive ion mode on a hybrid ion trap-Fourier transform ion cyclotron resonance mass spectrometer (Thermo Electron Corp., San Jose, CA). Nanoflow HPLC was performed using a similar approach to that described by Yi et al. (28.Yi E.C. Lee H. Aebersold R. Goodlett D.R. Rapid Commun. Mass Spectrom. 2003; 17: 2093-2098Crossref PubMed Scopus (96) Google Scholar). The electrospray voltage was applied via a liquid junction using a platinum wire inserted into a micro-tee union (Upchurch Scientific, Oak Harbor, WA). Ion source conditions were as follows: ESI voltage, 1.4 kV; capillary temperature, 200 °C; capillary voltage, 44 V; and tube lens voltage, 180 V. All other voltages were optimized using a tuning solution composed of caffeine (Sigma), MRFA (Bachem, King of Prussia, PA) and Ultramark 1621 (Lancaster Synthesis, Windham, NH). Injection waveforms for the LTQ-FT ion trap and ICR cell were kept on for all acquisitions. ICR resolution was set to 50,000 (m/z 400). ICR ion populations in the ICR cell were held at 1e6 and 5e5 for MS and MS/MS, respectively. For MS/MS, the precursor isolation width was set to 10 Da and the collision energy set to 40 and 55% for quintuply and quadruply charged precursor ions, respectively. Acetaminophen (APAP) Oxidation Catalyzed by CYP2E1—Assays of APAP oxidation metabolites were performed with slight modifications to a previously described procedure (12.Chen W. Koenigs L.L. Thompson S.J. Peter R.M. Rettie A.E. Trager W.F. Nelson S.D. Chem. Res. Toxicol. 1998; 11: 295-301Crossref PubMed Scopus (206) Google Scholar). Briefly, p" @default.
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• W2013396018 date "2006-07-01" @default.
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• W2013396018 title "Identification of the Interactions between Cytochrome P450 2E1 and Cytochrome b5 by Mass Spectrometry and Site-directed Mutagenesis" @default.
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