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• W2073224154 abstract "Liver toxicity following an overdose of acetaminophen is frequently considered a model for drug-induced hepatotoxicity. Extensive studies over many years have established that such toxicity is well correlated with liver protein arylation by acetaminophen metabolites. Identification of protein targets for covalent modifications is a challenging but necessary step in understanding how covalent binding could lead to liver toxicity. Previous approaches suffered from technical limitations, and thus over the last 10 years heroic efforts were required to determine the identity of only a few target proteins. We present a new mass spectrometry-based strategy for identification of all target proteins that now provides a comprehensive survey of the suite of liver proteins modified. After administration of radiolabeled acetaminophen to mice, the proteins in the liver tissue lysate were separated by two-dimensional polyacrylamide gel electrophoresis. In-gel digestion of the radiolabeled gel spots gave a set of tryptic peptides, which were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Interrogation of data bases based on experimentally determined molecular weights of peptides and product ion tags from postsource decay mass spectra was employed for the determination of the identities of modified liver proteins. Using this method, more than 20 new drug-labeled proteins have been identified. Liver toxicity following an overdose of acetaminophen is frequently considered a model for drug-induced hepatotoxicity. Extensive studies over many years have established that such toxicity is well correlated with liver protein arylation by acetaminophen metabolites. Identification of protein targets for covalent modifications is a challenging but necessary step in understanding how covalent binding could lead to liver toxicity. Previous approaches suffered from technical limitations, and thus over the last 10 years heroic efforts were required to determine the identity of only a few target proteins. We present a new mass spectrometry-based strategy for identification of all target proteins that now provides a comprehensive survey of the suite of liver proteins modified. After administration of radiolabeled acetaminophen to mice, the proteins in the liver tissue lysate were separated by two-dimensional polyacrylamide gel electrophoresis. In-gel digestion of the radiolabeled gel spots gave a set of tryptic peptides, which were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Interrogation of data bases based on experimentally determined molecular weights of peptides and product ion tags from postsource decay mass spectra was employed for the determination of the identities of modified liver proteins. Using this method, more than 20 new drug-labeled proteins have been identified. Acetaminophen is a widely used over-the-counter analgesic and antipyretic. It is therapeutically safe and has a high therapeutic index. It is commonly used as a substitute for aspirin because of its lower incidence of side effects. However, an overdose of acetaminophen may cause acute, often fatal, centrilobular liver necrosis in both humans and animals (1Hinson J.A. Pohl L.R. Monks T.J. Gillette J.R. Life Sci. 1981; 29: 107-116Crossref PubMed Scopus (157) Google Scholar). For more than 2 decades, evidence has mounted implicating the cytochrome P-450 metabolite,N-acetyl-p-benzoquinone imine (NAPQI), 1The abbreviations used are: NAPQI,N-acetyl-p-benzoquinone imine; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MS, mass spectrometry; PSD, postsource decay; EST, expressed sequence tag; GST, glutathione S-transferase. 1The abbreviations used are: NAPQI,N-acetyl-p-benzoquinone imine; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption ionization; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MS, mass spectrometry; PSD, postsource decay; EST, expressed sequence tag; GST, glutathione S-transferase. as the reactive intermediate responsible for the toxicity (2Harvison P.J. Guengerich F.P. Rashed M.S. Nelson S.D. Chem. Res. Toxicol. 1988; 1: 47-52Crossref PubMed Scopus (105) Google Scholar, 3Corcoran G.B. Mitchell J.R. Vaishnav Y.N. Horning E.C. Mol. Pharmacol. 1980; 18: 536-542PubMed Google Scholar, 4Dahlin D.C. Nelson S.D. J. Med. Chem. 1982; 25: 885-886Crossref PubMed Scopus (173) Google Scholar, 5Dahlin D.C. Miwa G.T. Lu A.Y. Nelson S.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1327-1331Crossref PubMed Google Scholar, 6Holme J.A. Dahlin D.C. Nelson S.D. Dybing E. Biochem. Pharmacol. 1984; 33: 401-406Crossref PubMed Scopus (109) Google Scholar, 7Miner D.J. Kissinger P.T. Biochem. Pharmacol. 1979; 28: 3285-3290Crossref PubMed Scopus (213) Google Scholar). At therapeutic doses, this metabolite is efficiently detoxified by reduced glutathione (GSH) to form an acetaminophen-glutathione conjugate (8Potter W.Z. Thorgeirsson S.S. Jollow D.J. Mitchell J.R. Pharmacology. 1974; 12: 129-143Crossref PubMed Scopus (265) Google Scholar). However, following toxic doses of acetaminophen, available glutathione pools are depleted, allowing reactive metabolites access to liver proteins, the putative mediators of the hepatotoxicity observed.As early as 1973, Jollow et al. (9Jollow D.J. Mitchell J.R. Potter W.Z. Davis D.C. Gillette J.R. Brodie B.B. J. Pharmacol. Exp. Ther. 1973; 187: 195-202PubMed Google Scholar) found that the drug-protein adducts accumulate in hepatocytes in the centrilobular region of the liver, wherein hepatocytes undergo extensive necrosis after acetaminophen administration. Moreover, pretreatment of animals with agents that either increase or decrease NAPQI formation yields parallel increases or decreases in binding to proteins in hepatocytes correlated with hepatocellular damage. This general association of the degree of protein covalent modification with severity of toxicity led Jollow and co-workers (9Jollow D.J. Mitchell J.R. Potter W.Z. Davis D.C. Gillette J.R. Brodie B.B. J. Pharmacol. Exp. Ther. 1973; 187: 195-202PubMed Google Scholar) to suggest that “acetaminophen-induced hepatic necrosis may be caused by the covalent binding of a chemically reactive metabolite to vital hepatic macromolecules.” This hypothesis has found support in more recent studies utilizing immunochemical analyses reported by Roberts et al. (10Roberts D.W. Bucci T.J. Benson R.W. Warbritton A.R. McRae T.A. Pumford N.R. Hinson J.A. Am. J. Pathol. 1991; 138: 359-371PubMed Google Scholar, 11Roberts D.W. Pumford N.R. Potter D.W. Benson R.W. Hinson J.A. J. Pharmacol. Exp. Ther. 1987; 241: 527-533PubMed Google Scholar) and Bartoloneet al. (12Bartolone J.B. Cohen S.D. Khairallah E.A. Fundam. Appl. Toxicol. 1989; 13: 859-862Crossref PubMed Scopus (21) Google Scholar, 13Bartolone J.B. Sparks K. Cohen S.D. Khairallah E.A. Biochem. Pharmacol. 1987; 36: 1193-1196Crossref PubMed Scopus (102) Google Scholar). Using Western immunoblotting to hepatic proteins separated using one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE), these workers demonstrated that a small number of proteins appeared susceptible to drug modification, which suggests considerable selectivity with respect to the particular proteins targeted (14Pumford N.R. Roberts D.W. Benson R.W. Hinson J.A. Biochem. Pharmacol. 1990; 40: 573-579Crossref PubMed Scopus (53) Google Scholar, 15Bartolone J.B. Beierschmitt W.P. Birge R.B. Hart S.G. Wyand S. Cohen S.D. Khairallah E.A. Toxicol. Appl. Pharmacol. 1989; 99: 240-249Crossref PubMed Scopus (68) Google Scholar). Investigations carried out using antibodies prepared from different haptens have revealed somewhat different patterns of gel bands that contain putative acetaminophen-protein adducts. These results indicate most extensive modification occurring in a band of proteins around 56 kDa (14Pumford N.R. Roberts D.W. Benson R.W. Hinson J.A. Biochem. Pharmacol. 1990; 40: 573-579Crossref PubMed Scopus (53) Google Scholar, 15Bartolone J.B. Beierschmitt W.P. Birge R.B. Hart S.G. Wyand S. Cohen S.D. Khairallah E.A. Toxicol. Appl. Pharmacol. 1989; 99: 240-249Crossref PubMed Scopus (68) Google Scholar, 16Bartolone J.B. Birge R.B. Sparks K. Cohen S.D. Khairallah E.A. Biochem. Pharmacol. 1988; 37: 4763-4774Crossref PubMed Scopus (83) Google Scholar).Although drug/protein adduct formation has long been thought to play a key role in acetaminophen-induced hepatotoxicity, little is known about the processes or mechanisms by which such modification(s) to particular target proteins might lead to liver cell necrosis. Throughout the last decade, several groups have expended considerable efforts in this regard, resulting in the identification of only a handful of the entire suite of hepatic proteins known to be targets of drug metabolite reactivity. These include selenium-binding protein (17Bartolone J.B. Birge R.B. Bulera S.J. Bruno M.K. Nishanian E.V. Cohen S.D. Khairallah E.A. Toxicol. Appl. Pharmacol. 1992; 113: 19-29Crossref PubMed Scopus (88) Google Scholar, 18Pumford N.R. Martin B.M. Hinson J.A. Biochem. Biophys. Res. Commun. 1992; 182: 1348-1355Crossref PubMed Scopus (99) Google Scholar), a subunit of glutamine synthetase (19Bulera S.J. Birge R.B. Cohen S.D. Khairallah E.A. Toxicol. Appl. Pharmacol. 1995; 134: 313-320Crossref PubMed Scopus (60) Google Scholar), N-10-formyltetrahydrofolate dehydrogenase (20Pumford N.R. Halmes N.C. Martin B.M. Cook R.J. Wagner C. Hinson J.A. J. Pharmacol. Exp. Ther. 1997; 280: 501-505PubMed Google Scholar), glutamate dehydrogenase (21Halmes N.C. Hinson J.A. Martin B.M. Pumford N.R. Chem. Res. Toxicol. 1996; 9: 541-546Crossref PubMed Scopus (59) Google Scholar), aldehyde dehydrogenase (22Landin J.S. Cohen S.D. Khairallah E.A. Toxicol. Appl. Pharmacol. 1996; 141: 299-307Crossref PubMed Google Scholar), lamin-A (23Hong M. Cohen S.D. Khairallah E.A. Toxicologist. 1994; 427Google Scholar), and carbamyl phosphate synthetase I (24Gupta S. Rogers L.K. Smith C.V. Toxicologist. 1995; 153Google Scholar). Because NAPQI is a very reactive metabolite that binds to numerous hepatic proteins in vivo, binding to any particular protein may be advantitious and does not constitute proof that such a component is a critical protein target contributing to acetaminophen-induced liver toxicity.At this juncture, there is a compelling need to gain a comprehensive knowledge of the entire suite of modified proteins together with the structural nature of their covalent adducts so that further experiments aimed at elucidation of the biochemical basis of compromised cell homeostasis may proceed.In this contribution, we wish to report the identities of all major hepatic proteins covalently modified after administration of a toxic dose of acetaminophen in the mouse. Using a combination of techniques including two-dimensional SDS-PAGE, fluorography, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we have identified 23 hepatic proteins. Having a knowledge of the entire suite of targets should yield new insights into the role of covalent drug binding in the pathogenesis of toxic doses of acetaminophen.EXPERIMENTAL PROCEDURESMaterialsAcetaminophen was purchased from Aldrich. [ring-U-14C]Acetaminophen (6.3 mCi/mmol) was obtained from Sigma. The radiochemical purity of [ring-U-14C]acetaminophen was greater than 96%, as determined by HPLC and 1H NMR on a 300-MHz GE instrument. Electrophoresis reagents were obtained either from Sigma or Amersham Pharmacia Biotech. HPLC grade solvents were obtained from Fisher.AnimalsB6C3F1 mice were obtained from Simonsen (Gilroy, CA). Prior to the administration of acetaminophen, mice were given phenobarbital as a 0.1% solution in their drinking water for 5 days, and food was withheld from all mice for 15 h. Both radiolabeled and nonlabeled acetaminophen were given as aqueous solutions (20 mg/ml) at doses of 350 mg/kg. The mice were killed by cervical dislocation 2 h after dosing. Gallbladders were removed before livers were subjected to the following procedures.Sample PreparationLivers were weighed and homogenized with a Duall glass type homogenizer (Kontes Glass Co., Vineland, NJ) in 8 × liver weight (g) ml of a solubilizing solution of 7 m urea, 2m thiourea, 4% CHAPS, 65 mm dithiothreitol, and pharmalyte, pH 3–10 (0.36 meq/ml, 1:50 by volume). Thiourea (2m) was added to the homogenizing buffer to increase the solubility of more hydrophobic proteins (25Rabilloud T. Adessi C. Giraudel A. Lunardi J. Electrophoresis. 1997; 18: 307-316Crossref PubMed Scopus (403) Google Scholar). The homogenate was centrifuged at either 10,000 or 100,000 × g for 30 min. The supernatant protein was removed and stored at −80 °C as 1-ml aliquots in 1.5-ml microcentrifuge tubes. The protein concentration was determined by the Bio-Rad protein assay, with bovine serum albumin as a standard.Covalent BindingCovalent binding of acetaminophen to proteins in liver homogenate was determined at 2.32 nmol/mg of proteins as described previously (26Rashed M.S. Myers T.G. Nelson S.D. Drug Metab. Dispos. 1990; 18: 765-770PubMed Google Scholar). Briefly, exhaustive washing was performed with cold methanol/diethylether (3:1, v/v) to remove reversibly bound drug until the radioactivity in the supernatant was less than the background level in two consecutive washes. The remaining protein pellet was dissolved in 0.25 m potassium hydroxide solution at 80 °C for 1 h. The clear solution was then subjected to scintillation counting.Two-dimensional Gel Electrophoresis and AutoradiographyElectrophoresis of mouse liver proteins was performed according to the manufacturer's instructions with the following modifications. Immobiline DryStrips (18 cm, pH 3–10 NL (nonlinear)) were used for isoelectric focusing on the MultiPhor II (Amersham Pharmacia Biotech). Protein samples (200 μl) prepared as described above, 200 ml of rehydration buffer (7 m urea, 2 m thiourea, 4% CHAPS, 65 mm dithiothreitol, pharmalyte pH 3–10 (0.36 meq/ml, 1:50 by volume)), and a trace of bromphenol blue) were placed in a rehydration tray (27Sanchez J.C. Rouge V. Pisteur M. Ravier F. Tonella L. Moosmayer M. Wilkins M.R. Hochstrasser D.F. Electrophoresis. 1997; 18: 324-327Crossref PubMed Scopus (283) Google Scholar) with the Immobiline DryStrips overnight. Proteins were focused at 20 °C according to the following voltage gradient program: 0–300 V, 2 h; 300–1000 V, 1 h; 1000–3500 V, 1 h; 3500 V, 15 h, using an EPS 3500 XL (Amersham Pharmacia Biotech) electrophoresis power supply. After a standard SDS equilibrating step, proteins were further separated by SDS-PAGE as described by Anderson (Ref. 28Anderson L. Two-dimensional Electrophoresis: Operation of the ISO-DALT System. 2nd Ed. Large Scale Biology Press, Rockville, MD1991Google Scholar). The separating gel was 11% light nongradient gel made with 30.8% DuracrylTM solution (Chelmsford, MA). The stacking gel contained 4% acrylamide/bis. The gels were run at 50 mA/gel for 9–10 h and then stained for 15 h in 0.1% Coomassie Blue R-250, 45% methanol, 10% acetic acid, 45% water. Gels were destained with 45% methanol, 10% acetic acid, 45% water. Spots containing radioactivity were detected by autoradiography as described in the operations manual (28Anderson L. Two-dimensional Electrophoresis: Operation of the ISO-DALT System. 2nd Ed. Large Scale Biology Press, Rockville, MD1991Google Scholar) with 8 × 10-inch Kodak X-Omat XAR-2 film.In-gel DigestionThe procedure of Rosenfeld et al. (29Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1126) Google Scholar), with slight modifications, was used to produce in-gel tryptic digest of all spots of interest.HPLC and DesaltingHPLC separation was performed according to Hall et al. (30Hall S.C. Smith D.M. Masiarz F.R. Soo V.W. Tran H.M. Epstein L.B. Burlingame A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1927-1931Crossref PubMed Scopus (87) Google Scholar). The salt content was removed by the following procedure. Briefly, samples were dried in a SpeedVac and redissolved in 10 μl of 1% acetonitrile, 1% trifluoroacetic acid, water. Using a 10-μl syringe, the reconstituted peptide solution was injected onto a LC Packings μ-guard column (300 μm inner diameter × 1 mm; 5-μm particle size; 300-Å, pore size) preequilibrated with 1% acetonitrile, 1% trifluoroacetic acid, water at a rate of no more than 5 μl/min. After washing off the salt with 3 × 10 μl of 1% acetonitrile, 1% trifluoroacetic acid, water at a rate of 10 μl/min, the peptides were eluted by injecting 10, 30, 50, and 90% acetonitrile in 1% trifluoroacetic acid, water (10 μl each in 1 min), consecutively. Eluents were collected, dried, and finally reconstituted in 10 μl of 2% trifluoroacetic acid, 50% acetonitrile, water.Mass SpectrometryMolecular Weight Measurements on Unseparated Tryptic DigestsMolecular weights of all peptides were determined by analyzing one-twentieth of the unseparated tryptic digests employing a matrix-assisted laser desorption (MALDI) delayed extraction reflectron time-of-flight instrument (PerSeptive Biosystems, Voyager Elite mass spectrometer, Framingham, MA) equipped with a nitrogen laser (337 mm), which has a typical mass resolution, M/dM, of 6000 (FWHM). Peptides were crystallized in a saturated solution of α-cyano-4-hydroxycinnamic acid prepared in 0.1% trifluoroacetic acid, 50% acetonitrile, 50% water. The monoisotopic (31Burlingame A.L. Carr S.A. Burlingame A.L. Carr S.A. Mass Spectrometry in the Biological Sciences. Humana, Totowa, NJ1996: 546-553Google Scholar) masses from all spectra recorded for a particular peptide are reported in this work. All MALDI spectra were externally calibrated by using a standard mixture of known peptides.Mass Spectrometric Determination of Partial Peptide SequenceAfter inspection of the MALDI mass spectra of the unseparated mixture of peptides from the spot digest, those components displaying the highest pseudomolecular ion abundance were selected for partial amino acid sequence determination. This was carried out by taking advantage of the inherent metastable fragmentation induced by deposition of excess internal energy during the laser desorption process by recording so-called postsource decay (PSD) mass spectra (32Rouse J.C. Yu W. Martin S.A. J. Am. Soc. Mass Spectrom. 1995; 6: 822-835Crossref PubMed Scopus (55) Google Scholar). Peptides with similar mass values were separated by microbore HPLC before carrying out pseudomolecular ion gating and PSD analysis. Most samples were subjected to a desalting step as described above to obtain higher quality mass spectra (improved signal versusnoise ratio can be achieved after such a clean-up step) that facilitate PSD experiments.Data Base SearchingMS-Fit and MS-Tag were used to perform data base searching. Both programs were developed by Clauser et al. in our group (University of California San Francisco Mass Spectrometry Facility) (33Clauser K.R. Baker P. Burlingame A.L. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12–16, 1996. 1996; : 365Google Scholar). 2Available on the World Wide Web athttp://rafael.ucsf.edu. MS-Fit is a typical peptide mass fingerprinting program, which compares the experimentally determined masses of tryptic peptides with the theoretical masses of all tryptic peptides that can be calculated from sequences of all proteins in the genomic data bases. All of the proteins (data base entries) matching the input data/parameters were listed in a simple ranking system in which data base entries with the least number of unmatched masses are ranked higher. MS-Tag is a sequence data base searching tool used to match fragment ion tag data contained in a user's tandem mass spectrum to a peptide sequence in an existing data base. Ideally, one would prefer to obtain a tandem mass spectrum with enough fragment ions from which a complete peptide sequence can be determined. In practice, samples such as peptides from in-gel digestion of a two-dimensional PAGE gel spot usually only yield a limited number of fragment ions by PSD, which prevents manual interpretation of these tandem mass spectra. However, each fragment ion is characteristic of the sequence of the peptide under analysis and adds a constraint to data base searching. Therefore, all ions present provide very high discriminating power for searching genomic data bases. MS-Tag integrates all of these constraints by considering each fragment ion and parent ion mass independently to match a single peptide sequence in a genomic data base, so the identity of the protein on a gel spot of interest is also determined. A variety of fragment ion types, as well as immonium ions and internal sequence ions, can be used by the MS-Tag algorithm. This not only facilitates the identification of peptides that are identical to sequences in the data base but also enables homology-tolerant searching to allow for a single mutation, cross-species substitution, sequence polymorphism, modified amino acid, or data base error. The NCBInr protein data base was searched first, and if no match was found, the dbEST DNA data base was further searched to determine the identity of the target protein or a protein homologous to the target protein.RESULTSTwo-dimensional Electrophoresis and AutoradiographySample application and rehydration of Immobiline DryStrips were combined in one step to ease the operation and, more importantly, to increase loading capacity without horizontal streaking and protein precipitation at the sample application point (27Sanchez J.C. Rouge V. Pisteur M. Ravier F. Tonella L. Moosmayer M. Wilkins M.R. Hochstrasser D.F. Electrophoresis. 1997; 18: 324-327Crossref PubMed Scopus (283) Google Scholar). Gels prepared from a single batch were found to be virtually identical, thus readily permitting inter-gel spot correlation. An example of a two-dimensional preparative gel of a whole liver homogenate stained with Coomassie Blue is depicted in Fig. 1 A. This homogenate was derived from the excised whole liver of a phenobarbital-induced B6C3F1 mouse after treatment with a toxic dose of14C-labeled acetaminophen. This gel was then exposed to film for 2 weeks, and the resulting autoradiogram is shown in Fig. 1 B. Twenty- seven major 14C-containing spots were revealed. Spot-to-spot comparison allowed in-gel digestions to be performed on analogous gels without radioactivity but after administration of the same amount of drug.In-gel Digestion and Mass SpectrometryThe strategy used for identification of acetaminophen targeted proteins is outlined in Fig. 2. Protein spots corresponding to the exposures on the radiogram (Fig. 1 B) were excised from several preparative gels, pooled, and then subjected to in-gel digestion procedures as described above. The number of peptides detected from the unseparated digest of a protein in a two-dimensional PAGE spot varied, since the peptide recovery depends on a variety of factors such as protein size, amino acid composition, hydrophobicity, etc. (35Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5072-5076Crossref PubMed Scopus (201) Google Scholar).Figure 2Strategy for identifying proteins targeted by acetaminophen employing two-dimensional PAGE, MALDI-MS.View Large Image Figure ViewerDownload (PPT)Data Base SearchingData base interrogation based on the experimentally determined values of peptides (peptide mass map) from in-gel digestion was carried out routinely on all gel spot digests using MS-Fit. This strategy is known not to be a rigorously definitive criterion for establishing protein identity by computer-based matching of data base entries (35Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5072-5076Crossref PubMed Scopus (201) Google Scholar).To circumvent these known difficulties, partial peptide sequence information was obtained based on recording PSD mass spectra, and MS-Tag was used for data base interrogation using the sequence and composition information obtained (33Clauser K.R. Baker P. Burlingame A.L. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12–16, 1996. 1996; : 365Google Scholar).2 A typical PSD spectrum does not provide complete sequence ions (a, b, c, x, y, and z ions) of a particular peptide, although a, b, and y ions in the lower mass region are abundant. Internal ions with lower masses (<600 Da) and immonium ions are also evident. Data base searching with MS-Tag uses all of this information together with the mass of the peptide to match the theoretical counterparts calculated from all protein sequences in the data base and to determine the identity of the protein. MS-Tag searching based on one PSD spectrum is usually specific enough to identify a unique protein if the protein is in the data base. Therefore, multiple proteins in mixtures may be identified with confidence. To further increase the confidence of an unambiguous identification, often PSD spectra of several additional peptides were obtained for one gel spot. Finally, the identification can be verified by checking how many of the remaining peptide masses measured by MALDI-MS match the theoretical masses of tryptic peptides from the protein matched.Table I summarizes peptide mass data, sequences determined or attributed by mass, and data base searching results for all protein spots studied. Peptide masses not analyzed by PSD were attributed to a unique sequence by matching the mass to a tryptic peptide from the matched protein (masses of peptides from nonspecific cleavages are not considered). Since the peptide mass measurements were externally calibrated, the mass differences between experimental and theoretical values are different for peptides from different proteins. However, they are very similar for peptides from the same protein (spot) and proportional to the masses of peptides.Table ISummary of data obtained for 30 acetaminophen-modified mouse liver proteins from 30 two-dimensional PAGE gel spotsSpot No.MALDI massΔaThe difference between the measured mass and calculated mass (monoisotopic).Peptide sequence determined by PSD and consistent with massbSequence determined by PSD is underlined. Residues before/after peptide are in parentheses. Cam, acrylamide-modified Cys; Cim, iodoacetamide-modified Cys; Ac-, acetylated N terminus; Met-ox, methionine sulfoxide; pyro-Glu, pyroglutamine at the N terminus; u, selenocysteine; X, carbamidomethylated selenocysteine; SBP, selenium-binding protein; SBLP, selenium-binding liver protein; AP56, acetaminophen-binding protein.Protein identified (NCBI accession no.); percentage of the protein covered molecular massSubcellular locationDaDa1992.37−0.15(K)EPFTFPVR(G)Life Technologies mouse embryo 8 5dpc Mus musculus cDNA clone (dbEST: 2049563); 46%; 12,107.3 DaUnknown791.29−0.09(R)GICimGQTR(I)1681.80−0.19(R)LAEVGGVPYLLPLVNK(K)2292.79−0.31(K)GLTDNFADVQVSVVDCimPDLTK(E)932.321313.44817.30, 1114.48, 1128.44, 1153.41, 1327.44, 1371.54, 1393.42, 1953.71, 2221.82, 2433.95, 2586.05cMasses neither identified nor attributed.21101.38−0.24(K)AHPLFTFLR(N)Glutathione peroxidase (984747); 26%; 22,292.5 DaCytoplasmic mitochondrial1382.42−0.34(R)PLTGGEPVSLGSLR(G)1542.43−0.36(R)LSAAAQSTVYAFSAR(P)1667.60−0.37(K)VLLIENVASLuGTTIR(D)1957.57−0.41(K)YVRPGGGFEPNFTLFEK(C)1807.51−0.42(K)VLLIENVASLXGTTIR(D)1155.39−0.26(K)FLVGPDGVPVR(R)1311.46−0.29(K)FLVGPDGVPVRR(Y)31206.43−0.23(K)HLSVNDLPVGR(S)Housekeeping protein (126986); (15%); 28,127.2 DaMitochondrial1476.33−0.48(R)DYGVLESAGIALR(G)1943.89−0.17(K)pyro-GluISRDYGVLLESAGIALR(G)1960.75−0.33(K)QISRDYGVLLESAGIALR(G)2458.90−0.48(R)GLFIIDPNKVVKHLSVNDLPVGR(S)1794.552720.722748.33cMasses neither identified nor attributed.41858.39+0.23(R)LAGLLKPGGHLVTLVTLR(F)ThioetherS-methyltransferase (731019); 24%; 29,460.0 Da (1083533); 24%; 29,397.9 DaCytoplasmic or microsomal1106.71+0.16(R)FQHYMYGPK(K)1122.67+0.13(R)FQHYMet-oxVGPK(K)2032.30+0.24(R)EIIVTDYTPQNLQELQK(W)2447.48+0.30(K)DYLTTYYSFHSGPVAEQEIVK(F)1262.88cMasses neither identified nor attributed.51464.41−0.22(K)EEMDHSVSPFMR(K)Mouse NML M. musculus cDNA clone (dbEST: 1875803); 72%; homologous to rat Aryl sulfotransferase of 16,310.8 DaCytoplasmic891.44−0.01(K)SWWEKR(K)929.35−0.08(K)FEEDYVK(K)1070.57−0.00(K)DIPEEILNK(I)1227.59−0.01(K)NQFTVAQYEK(F)1311.64−0.04(K)ILYHSSFSVMK(E)1327.66−0.01(K)ILYHSSFSVMK(E)1334.60−0.01(R)ILYLFYEDMK(E)1418.16−0.43(K)ENPSANYTTMet-oxMet-oxK(E)1480.41−0.21(K)EEMDHSVSPFMR(K)-1Met-ox1802.85−0.05(R)ILYLFYEDMKENPK(C)1868.86−0.02(K)FMAGQVSFGPWYDHVK(S)1884.85−0.02(K)FMet-oxAGQVSFGPWYDHVK(S)2137.95−0.06(K)NQFTVAQYEKFEEDYVK(K)2266.05−0.05(K)NQFTVAQYEKFEEDYVKK(M)2832.18−0.03(K)ENPSANYTTMMKEEMDHSVSPFMR(K)734.451652.891711.80, 1723.73, 1775.80dGlu1 to Asp1., 2325.1561067.21−0.34(R)AAPFTLEYR(V)Homologous to bovine inorganic pyrophosphatase (585322); 18%; 32,844.4 Da1327.23−0.45(K)DdGlu1 to Asp1.VFHMVVEVPR(W)1694.33−0.56(R)LKPGYLEATVDWFR(R)1114.24−0.35(R)YVANLFPYK(G)1849.31−1.68(K)RLKPGYLEATVDWFR(R)1938.27−0.65(K)VPDGKPENEFAFNAEFK(D)2229.37−0.71(R)YKVPDGKPENEFAFNAEFK(D)940.071281.102088.27, 2431.38cMasses neither identified nor attributed.71156.41−0.25(K)LVIIEGDLER(T)Tropomyosin 5, cytoskeletal type (136097); 27%; 29,220.8 DaCytoskeletal1243.39−0.26(R)IQLVEEELDR(A)1284.48−0.27(R)KLVIIEGDLER(T)1642.47−0.33(K)IQVLQQQADDAEER(A)894.29−0.18(R)KYEEVAR(K) or (K)YEEVARK(L)940.25−0.24(K)HIAEEADR(K)1316.37−0.27(R)EQAEAEVASLNR(R)1399.41−0.34(R)RIQLVEEELDR(A)1472.23−0.51(R)EQAEAEVA" @default.
• W2073224154 created "2016-06-24" @default.
• W2073224154 creator A5031483671 @default.
• W2073224154 creator A5068865174 @default.
• W2073224154 creator A5075960682 @default.
• W2073224154 date "1998-07-01" @default.
• W2073224154 modified "2023-10-15" @default.
• W2073224154 title "Identification of the Hepatic Protein Targets of Reactive Metabolites of Acetaminophen in Vivoin Mice Using Two-dimensional Gel Electrophoresis and Mass Spectrometry" @default.
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• W2073224154 doi "https://doi.org/10.1074/jbc.273.28.17940" @default.
• W2073224154 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9651401" @default.
• W2073224154 hasPublicationYear "1998" @default.

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