Attomole Detection of in Vivo Protein Targets of Benzene in Mice
2002; Elsevier BV; Volume: 1; Issue: 11 Linguagem: Inglês
10.1074/mcp.m200067-mcp200
ISSN1535-9484
AutoresKatherine Williams, Tonya A. Carver, JJ L. Miranda, Antti Kautiainen, John S. Vogel, Karen H. Dingley, Michael A. Baldwin, Kenneth W. Turteltaub, Alma L. Burlingame,
Tópico(s)Biotin and Related Studies
ResumoModified proteins were detected in liver and bone marrow of mice following treatment with [14C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable 14C/13C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of 14C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of 14C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. 14C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene. Modified proteins were detected in liver and bone marrow of mice following treatment with [14C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable 14C/13C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of 14C. A number of proteins were identified by in-gel proteolysis and conventional mass spectrometric methods with the low molecular weight proteins identified including hemoglobin and several histones. The incorporation of 14C was largely proportional to the density of gel staining, giving little evidence that these proteins were specific targets for selective labeling. This was also true for individual histones subfractionated with Triton-acid-urea gels. A representative histone, H4, was isolated and digested with endopeptidase Asp-N, and the resulting peptides were separated by high performance liquid chromatography. 14C levels in collected fractions were determined, and the peptides were identified by conventional mass spectrometry. The modifications were distributed throughout the protein, and no particular amino acids or groups of amino acids were identified as selective targets. Thus chemical attack by one or more benzene metabolites upon histones was identified and confirmed, but the resulting modifications appeared to be largely nonspecific. This implies high reactivity toward proteins, enabling such attack to occur at multiple sites within multiple targets. It is not known to what extent, if any, the modification of the core histones may contribute to the carcinogenicity of benzene. Human exposure to benzene occurs both occupationally in the chemical and fuel industry and environmentally through automobile exhaust, gasoline, and cigarette smoke (1.Wallace L.A. The exposure of the general population to benzene.Environ. Health Perspect. 1989; 82: 165-169Google Scholar). Chronic exposure to benzene is known to cause aplastic anemia and an increased risk of acute myelogenous leukemia in humans (2.Goldstein B.D.J. Benzene toxicity: a critical evaluation: introduction.Toxicol. Environ. Health. 1977; 2: 69-105Google Scholar, 3.Rinsky R.A. Smith A.B. Hornung R.F. Filloon T.G. Young R.J. Okun A.H. Landrigan P.J. Benzene and leukemia. An epidemiologic risk assessment.N. Engl. J. Med. 1987; 316: 1044-1050Google Scholar). High exposures result in toxic responses in rodent bone marrow, including aplastic anemia, micronuclei, leukocytopenia, and hyperplasia (4.Huff J.E. Haseman J.K. DeMarini D.M. Eustis S. Maronpot R.R. Peters A.C. Pershing R.L. Chrisp C.C. Jacobs A.C. Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.Environ. Health Perspect. 1989; 82: 125-163Google Scholar, 5.Snyder C.A. Goldstein B.D. Sellakumar A. Wolman S.R. Bromberg I. Erlichman M.N. Laskin S. Hematotoxicity of inhaled benzene to Sprague-Dawley rats and AKR mice at 300 ppm.J. Toxicol. Environ. Health. 1978; 4: 605-619Google Scholar, 6.MacEachern L. Snyder R. Laskin D.L. Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice.Toxicol. Appl. Pharmacol. 1992; 117: 147-154Google Scholar). Long term animal studies show that benzene causes tumors at multiple sites in mice and rats (4.Huff J.E. Haseman J.K. DeMarini D.M. Eustis S. Maronpot R.R. Peters A.C. Pershing R.L. Chrisp C.C. Jacobs A.C. Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.Environ. Health Perspect. 1989; 82: 125-163Google Scholar). However, the effects of the numerous electrophilic metabolites of benzene and molecular processes underlying hematotoxicity and leukemogenesis remain unclear.Benzene is oxidized in the liver by cytochrome P4502E1 to form benzene oxide (7.Snyder R. Witz G. Goldstein B.D. The toxicology of benzene.Environ. Health Perspect. 1993; 100: 293-306Google Scholar, 8.Kalf G.F. Recent advances in the metabolism and toxicity of benzene.CRC Crit. Rev. Toxicol. 1987; 18: 141-159Google Scholar, 9.Lovern M.R. Turner M.J. Meyer M. Kedderis G.L. Bechtold W.E. Schlosser P.M. Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes.Carcinogenesis. 1997; 18: 1695-1700Google Scholar), which gives rise to phenol, catechol, hydroquinone, 1,2,4-benzenetriol, and ring-opened metabolites such as trans-trans-muconic acid (10.Cooper K.R. Snyder R. Askoy M. Benzene Carcinogenicity. CRC Press, Boca Raton, FL1988: 33-58Google Scholar, 11.Seaton M.J. Schlosser P.M. 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Peroxidase-dependent metabolism of benzene's phenolic metabolites and its potential role in benzene toxicity and carcinogenicity.Environ. Health Perspect. 1989; 82: 23-29Google Scholar). Various combinations of benzene metabolites can exert a synergistic effect on multiple cellular targets leading to increased toxicity (19.Eastmond D.A. Smith M.T. Irons R.D. An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure.Toxicol. Appl. Pharmacol. 1987; 91: 85-95Google Scholar, 20.Barale R. Marrazzini A. Betti C. Vangelisti V. Loprieno N. Barrai I. Genotoxicity of two metabolites of benzene: phenol and hydroquinone show strong synergistic effects in vivo.Mutat. Res. 1990; 244: 15-20Google Scholar, 21.Guy R.L. Hu P. Witz G. Goldstein B.D. Depression of iron uptake into erythrocytes in mice by treatment with the combined benzene metabolites p-benzoquinone, muconaldehyde and hydroquinone.J. Appl. Toxicol. 1991; 11: 443-446Google Scholar). Bone marrow is a complex tissue containing hematopoietic stem cells and stromal cells, both of which are potential targets of benzene and its metabolites. There are likely multiple cellular and molecular targets of the metabolites in the bone marrow compartment that are involved in hematotoxicity and carcinogenicity (22.Ross D. Metabolic basis of benzene toxicity.Eur. J. Haematol. Suppl. 1996; 60: 111-118Google Scholar). Developments in the assessment of risks associated with exposure to benzene have been reviewed recently (23.Zeise L. McDonald T.A. California perspective on the assessment of benzene toxicological risks.J. Toxicol. Environ. Health Part A. 2000; 61: 479-483Google Scholar, 24.Kacew S. Lemaire I. Recent developments in benzene risk assessment.J. Toxicol. Environ. Health Part A. 2000; 61: 485-498Google Scholar).Benzene is known to cause aneuploidy but is not a strong DNA binding agent and is only weakly mutagenic (25.Creek M.R. Mani C. Vogel J.S. Turteltaub K.W. Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice.Carcinogenesis. 1997; 18: 2421-2427Google Scholar, 26.Dean B.J. Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols.Mutat. Res. 1985; 154: 153-181Google Scholar). We previously determined that [14C]benzene, administered at extremely low doses in mice, bound to proteins at a higher ratio compared with DNA in the target organ, bone marrow (25.Creek M.R. Mani C. Vogel J.S. Turteltaub K.W. Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice.Carcinogenesis. 1997; 18: 2421-2427Google Scholar, 27.Mani C. Freeman S. Nelson D.O. Vogel J.S. Turteltaub K.W. Species and strain comparisons in the macromolecular binding of extremely low doses of [14C]benzene in rodents, using accelerator mass spectrometry.Toxicol. Appl. Pharmacol. 1999; 159: 83-90Google Scholar). Elevated levels of benzene oxide and hydroquinone adducts of hemoglobin and albumin have been observed in workers subject to benzene exposure (28.Yeowell-O'Connell K. Rothman N. Smith M.T. Hayes R.B. Li G. Waidyanatha S. Dosemici M. Zhang L. Yin S. Titenko-Holland N. Rappaport S.M. Hemoglobin and albumin adducts of benzene oxide among workers exposed to high levels of benzene.Carcinogenesis. 1998; 19: 1565-1571Google Scholar, 29.Yeowell-O'Connell K. Rothman N. Waidyanatha S. Smith M.T. Hayes R.B. Li G. Bechtold W.E. Dosemeci M. Zhang L. Yin S. Rappaport S.M. Protein adducts of 1,4-benzoquinone and benzene oxide among smokers and nonsmokers exposed to benzene in China.Cancer Epidemiol. Biomark. Prev. 2001; 10: 831-838Google Scholar). It could be valuable to identify any other protein targets of benzene metabolites in the bone marrow to further our understanding of benzene-induced leukemogenesis.Accelerator mass spectrometry (AMS) 1The abbreviations used are: AMS, accelerator mass spectrometry; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TAU, Triton-acetic acid-urea 1The abbreviations used are: AMS, accelerator mass spectrometry; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TAU, Triton-acetic acid-urea provides an extremely sensitive method for the detection of low level modifications of proteins (30.Vogel J.S. Grant P.B. Bucholz B.A. Dingley K. Turteltaub K.W. Attomole quantitation of protein separations with accelerator mass spectrometry.Electrophoresis. 2001; 22: 2037-2045Google Scholar). We have utilized this technique to develop a powerful strategy for identification of target proteins of isotopically labeled xenobiotics in vivo, combining AMS with high resolution two-dimensional gel electrophoresis, Triton-acid-urea gels, and HPLC to separate complex protein mixtures and biological mass spectrometry (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and LC-ESI-MS) to identify polypeptides bearing 14C modifications. In addition to confirming previous reports of hemoglobin as a target for modification by benzene (30.Vogel J.S. Grant P.B. Bucholz B.A. Dingley K. Turteltaub K.W. Attomole quantitation of protein separations with accelerator mass spectrometry.Electrophoresis. 2001; 22: 2037-2045Google Scholar, 31.Ramagli L.S. Rodriguez L.V. Quantifying protein in 2-D PAGE solubilization buffers.Electrophoresis. 1985; 6: 559-563Google Scholar), we also report the identification of the core histones as targets for [14C]benzene metabolites detected at attomole levels in microgram amounts of mouse bone marrow protein.EXPERIMENTAL PROCEDURESChemicals—[U-14C]Benzene (specific activity, 58.2 mCi/mmol, corresponding to approximately one atom of 14C per benzene molecule) was obtained from Sigma. Radiopurity was found to be >99% by HPLC. The dosing solution was prepared in filter-sterilized corn oil by dilution of the stock [14C]benzene. The dosing solution concentration was verified by liquid scintillation counting. All chemicals were analytical or electrophoresis grade and obtained from Amersham Biosciences or Sigma.Animals—These investigations were conducted under established federal regulations for the care and use of laboratory animals and were approved by the Lawrence Livermore National Laboratory Animal Care Committee. Male B6C3F1 mice, each weighing 22–25 g, were purchased from Charles River Laboratory and allowed to acclimate in an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility for a minimum of 1 week before use. Animals were housed three to a cage in filter top, polycarbonate cages with hardwood chip bedding. They were maintained on a 12-h light/dark cycle at ≈22 °C and received laboratory chow and water ad libitum. Each animal received a single dose of corn oil (controls) or corn oil containing [14C]benzene via intraperitoneal injection. Each dosing stock was verified by liquid scintillation counting prior to administration. Two dosage regimens were used: 155 μg/kg of body weight, sacrificed at 1.5 h post-treatment, hereafter referred to as "low dose"; and 800 μg/kg of body weight, sacrificed at 18 h post-treatment, referred to as "high dose." All mice were euthanized by CO2 asphyxiation. The 18-h time point was selected based on previous time course data (25.Creek M.R. Mani C. Vogel J.S. Turteltaub K.W. Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice.Carcinogenesis. 1997; 18: 2421-2427Google Scholar, 27.Mani C. Freeman S. Nelson D.O. Vogel J.S. Turteltaub K.W. Species and strain comparisons in the macromolecular binding of extremely low doses of [14C]benzene in rodents, using accelerator mass spectrometry.Toxicol. Appl. Pharmacol. 1999; 159: 83-90Google Scholar) showing maximum adduct levels in the bone marrow at this time point.Sample Preparation—The humerus and femur bones were dissected from each mouse using disposable scalpels and forceps (those from the control mice being collected first) to ensure no 14C cross-contamination occurred. Tissues were not perfused to remove residual blood. The marrow was flushed from the bone shaft with 2 ml of phosphate-buffered saline, pH 7.4 using a disposable 26-gauge needle. Bone marrow from each treatment group was pooled, and the cells were washed three times in phosphate-buffered saline and centrifuged at 1000 × g. Cells were lysed in 9 m urea, 4% CHAPS, 65 mm dithiothreitol, 2% ampholytes (Pharmalyte 3.Rinsky R.A. Smith A.B. Hornung R.F. Filloon T.G. Young R.J. Okun A.H. Landrigan P.J. Benzene and leukemia. An epidemiologic risk assessment.N. Engl. J. Med. 1987; 316: 1044-1050Google Scholar, 4.Huff J.E. Haseman J.K. DeMarini D.M. Eustis S. Maronpot R.R. Peters A.C. Pershing R.L. Chrisp C.C. Jacobs A.C. Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.Environ. Health Perspect. 1989; 82: 125-163Google Scholar, 5.Snyder C.A. Goldstein B.D. Sellakumar A. Wolman S.R. Bromberg I. Erlichman M.N. Laskin S. Hematotoxicity of inhaled benzene to Sprague-Dawley rats and AKR mice at 300 ppm.J. Toxicol. Environ. Health. 1978; 4: 605-619Google Scholar, 6.MacEachern L. Snyder R. Laskin D.L. Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice.Toxicol. Appl. Pharmacol. 1992; 117: 147-154Google Scholar, 7.Snyder R. Witz G. Goldstein B.D. The toxicology of benzene.Environ. Health Perspect. 1993; 100: 293-306Google Scholar, 8.Kalf G.F. Recent advances in the metabolism and toxicity of benzene.CRC Crit. Rev. Toxicol. 1987; 18: 141-159Google Scholar, 9.Lovern M.R. Turner M.J. Meyer M. Kedderis G.L. Bechtold W.E. Schlosser P.M. Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes.Carcinogenesis. 1997; 18: 1695-1700Google Scholar, 10.Cooper K.R. Snyder R. Askoy M. Benzene Carcinogenicity. CRC Press, Boca Raton, FL1988: 33-58Google Scholar), 10 mm spermine, 40 mm Tris base and then centrifuged at 450,000 × g for 10 min at 22 °C in a Beckman TL100 tabletop ultracentrifuge to remove DNA. Protein concentrations in the supernatants were determined by a modified Bradford assay (31.Ramagli L.S. Rodriguez L.V. Quantifying protein in 2-D PAGE solubilization buffers.Electrophoresis. 1985; 6: 559-563Google Scholar) using the Bio-Rad protein assay kit and ovalbumin as a standard. Samples were stored at −80 °C prior to electrophoresis. Livers were dissected, cut into small pieces, and washed twice in 20 mm Tris-HCl, pH 7.4. The tissue was homogenized in 3 volumes of the same buffer and centrifuged at 9000 × g for 20 min. The supernatant was lysed with 2 volumes of sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.025% bromphenol blue), boiled for 5 min, and stored at −20 °C for at least 30 min. For histone purification, the acid-soluble histone fraction of bone marrow was precipitated with acetone (32.Yoshida M. Kijima M. Akita M. Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A.J. Biol. Chem. 1990; 265: 17174-17179Google Scholar, 33.Cousens L.S. Gallwitz D. Alberts B.M. Different accessibilities in chromatin to histone acetylase.J. Biol. Chem. 1979; 254: 1716-1723Google Scholar) and dissolved in H2O.One-dimensional Electrophoresis—Proteins were separated on 15% polyacrylamide gels and stained with 1% Coomassie Brilliant Blue in 50% methanol, 10% acetic acid. Triton-acetic acid-urea (TAU) gels (15% acrylamide) were run according to Waterborg (34.Waterborg J.H. Walker J.M. The Protein Protocols Handbook. Humana Press, Totowa, NJ1996: 91-100Google Scholar). Densitometry was carried out using ImageMaster (Amersham Biosciences).Two-dimensional Electrophoresis—Bone marrow proteins (100 μg) were separated by non-equilibrating pH gradient gel electrophoresis (35.O'Farrell P.Z. Goodman H.M. O'Farrell P.H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell. 1977; 12: 1133-1141Google Scholar) in 15-cm rod gels containing carrier ampholytes (pH 3.Rinsky R.A. Smith A.B. Hornung R.F. Filloon T.G. Young R.J. Okun A.H. Landrigan P.J. Benzene and leukemia. An epidemiologic risk assessment.N. Engl. J. Med. 1987; 316: 1044-1050Google Scholar, 4.Huff J.E. Haseman J.K. DeMarini D.M. Eustis S. Maronpot R.R. Peters A.C. Pershing R.L. Chrisp C.C. Jacobs A.C. Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.Environ. Health Perspect. 1989; 82: 125-163Google Scholar, 5.Snyder C.A. Goldstein B.D. Sellakumar A. Wolman S.R. Bromberg I. Erlichman M.N. Laskin S. Hematotoxicity of inhaled benzene to Sprague-Dawley rats and AKR mice at 300 ppm.J. Toxicol. Environ. Health. 1978; 4: 605-619Google Scholar, 6.MacEachern L. Snyder R. Laskin D.L. Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice.Toxicol. Appl. Pharmacol. 1992; 117: 147-154Google Scholar, 7.Snyder R. Witz G. Goldstein B.D. The toxicology of benzene.Environ. Health Perspect. 1993; 100: 293-306Google Scholar, 8.Kalf G.F. Recent advances in the metabolism and toxicity of benzene.CRC Crit. Rev. Toxicol. 1987; 18: 141-159Google Scholar, 9.Lovern M.R. Turner M.J. Meyer M. Kedderis G.L. Bechtold W.E. Schlosser P.M. Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes.Carcinogenesis. 1997; 18: 1695-1700Google Scholar, 10.Cooper K.R. Snyder R. Askoy M. Benzene Carcinogenicity. CRC Press, Boca Raton, FL1988: 33-58Google Scholar) for 2400 V-h. The gels were equilibrated for 10 min in 2% SDS, 5% dithiothreitol, 10% glycerol, trace bromphenol blue, and 1.5% Tris-HCl, pH 6.8 (36.O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar) prior to SDS-PAGE in 15% polyacrylamide gels. Proteins were visualized by silver staining (37.Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). Four identical two-dimensional gels were run for the 14C-containing bone marrow, and four gels were run for the controls. Each of the labeled gels and three unlabeled gels were analyzed separately by AMS. A fourth control gel was used for in-gel digestion, peptide extraction, and identification by MALDI-MS and LC-ESI-MS.HPLC—Reversed phase HPLC was performed on a Beckman Gold system. Core histones were separated based on previously published methods (32.Yoshida M. Kijima M. Akita M. Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A.J. Biol. Chem. 1990; 265: 17174-17179Google Scholar, 33.Cousens L.S. Gallwitz D. Alberts B.M. Different accessibilities in chromatin to histone acetylase.J. Biol. Chem. 1979; 254: 1716-1723Google Scholar) using a 180-μm × 15-cm C4 column (Vydac) and eluted with a 38–55% B linear gradient over 120 min (Buffer A: 0.1% heptafluorobutyric acid in H2O; buffer B, 0.08% heptafluorobutyric acid in acetonitrile). HPLC separation of AspN-digested peptides of histone H4 was carried out using a C18 column (180 μm × 15 cm) (LC Packings, San Francisco, CA) with an Applied Biosystems 140B syringe pump.AMS—Disposable materials were used for any item that might come in contact with a sample to prevent cross-contamination between samples. Gel spots were excised with disposable plastic drinking straws and placed in 6- × 50-mm quartz tubes. Tributyrin was added as a carrier to HPLC fractions. Samples were dried by vacuum centrifugation, and the dried samples were reduced to graphite using published protocols (38.Vogel J.S. Rapid production of graphite without contamination for biomedical AMS.Radiocarbon. 1992; 3: 344-350Google Scholar). For [14C]benzene analysis, we measured 14C relative to 13C and normalized to the ratio of 1950 AD carbon using the Australian National University sugar reference standard (39.Turteltaub K.W. Vogel J.S. Burlingame A.L. Carr S.A. Mass Spectrometry in the Biological Sciences. Humana Press, Totowa, NJ1996: 477-495Google Scholar). The carbon contents of the samples was determined using a C:N:S analyzer (Carlo-Erba NA1500, series 2). Hemoglobin was determined to have a carbon content of 50.38%.Enzymatic Digestion of Proteins—The procedure of Clauser et al. (40.Clauser K.R. Hall S.C. Smith D.M. Webb J.W. Andrews L.E. Tran H.M. Epstein L.B. Burlingame A.L. Rapid mass spectrometric sequencing and peptide mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5072-5076Google Scholar) was used with minor modifications for the tryptic digestion of proteins in polyacrylamide gels. Each spot was excised, diced, and vortexed three times for 10 min in 50% acetonitrile, 25 mm ammonium bicarbonate. The gel pieces were dried in a Speedvac, resuspended in 25 mm ammonium bicarbonate with 12.5 ng/μl trypsin, and digested overnight at 37 °C. The peptides were extracted by vortexing three times for 10 min in 50% acetonitrile, 5% trifluoroacetic acid. Extracts were pooled, concentrated in a Speedvac to ∼1–2 μl, and dissolved in 10 μl of 50% acetonitrile, 5% trifluoroacetic acid. HPLC-separated histone H4 was digested with AspN for 4 h in 50 mm sodium phosphate, pH 8.0 at 37 °C.Mass Spectrometry—The molecular masses of peptides were determined by analyzing the unseparated tryptic digests using one of three mass spectrometric techniques. Routine peptide analysis utilized a MALDI-TOF DE STR mass spectrometer (Applied Biosystems, Framingham, MA) operated in reflectron mode. One-tenth of each sample digest was mixed in a 1:1 ratio with 33 mm α-cyano-4-hydroxycinnamic acid solution (Agilent, Palo Alto, CA). Spectra were internally calibrated using known trypsin autolysis products (41.Vestling M.M. Murphy C.M. Fenselau C. Recognition of trypsin autolysis products by high-performance liquid chromatography and mass spectrometry.Anal. Chem. 1990; 62: 2391-2394Google Scholar). Selected ions were subjected to postsource decay analysis (42.Spengler B. Kirsch D. Kaufmann R. Jaeger E. Peptide sequencing by matrix-assisted laser-desorption mass spectrometry.Rapid Commun. Mass Spectrom. 1992; 6: 105-108Google Scholar, 43.Yu W. Vath J.E. Huberty M.C. Martin S.A. Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry.Anal. Chem. 1993; 65: 3015-3023Google Scholar). High energy positive ion collision-induced dissociation mass spectra were acquired on a Kratos Analytical Instruments Concept IIHH four-sector tandem mass spectrometer (Kratos, Manchester, UK) (44.Walls F.C. Baldwin M.A. Falick A.M. Gibson B.W. Kaur S. Maltby D.A. Gillece-Castro B.L. Medzihradszky K.F. Evans S. Burlingame A.L. Burlingame A.L. McCloskey J.A. Biological Mass Spectrometry. Elsevier, New York1990: 285-314Google Scholar) equipped with a continuous flow, liquid inlet probe for liquid secondary ionization and a scanning charge-coupled device array detector. For HPLC-MS, a capillary HPLC system (ABI, Foster City, CA) was interfaced directly to a Mariner orthogonal acceleration time-of-flight mass spectrometer using electrospray ionization (Applied Biosystems). The unseparated digests were diluted in water and injected directly into a 180-μm × 15-cm capillary C18 column (LC Packings). Online HPLC-MS was performed using a formic acid/ethanol/propanol solvent system (45.Feldhoff R. Villafranca J.J. Techniques in Protein Chemistry II. Academic Press, Inc., San Diego, CA1991: 55-63Google Scholar) with a gradient of 5–60% solvent B in 2 h. A cone voltage setting of 175 V was used for collision-induced dissociation, giving rise to fragment ion peaks. Peptide masses and fragment ion masses measured by either MALDI-MS or LC-ESI-MS were analyzed with the MS-Fit and MS-Tag programs in ProteinProspector (prospector.ucsf.edu) (46.Clauser, K. R., Baker, P., and Burlingame, A. L. (1996) in Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12–16, 1996, p. 365, American Society for Mass Spectrometry, Santa Fe, NMGoogle Scholar). Mass accuracy in both MALDI-MS (internally calibrated) and ESI-MS was generally 50 ppm or better.RESULTSIdentificaton of 14C in Proteins from SDS-PAGE—AMS was used to determine the 14C incorporation in proteins separated by one-dimensional SDS-PAGE with Coomassie Blue staining throughout the molecular mass range up to 120 kDa in liver (low dose only) and bone marrow (low dose and high dose). In the liver, the highest levels of the heavy isotope were found in the protein fraction corresponding to the molecular mass range of 45–55 kDa (Fig. 1, upper panel). By contrast, in bone marrow the distribution of 14C proteins for both low and high dose showed the highest levels of incorporation in a band of 12–15 kDa. The distribution of 14C throughout the molecular mass range was similar for both doses of benzene in the bone marrow. It was noted that all regions of the gels showed significant levels of isotope incorporation compared with naturally occurring levels in undosed controls, suggesting that at least a
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