Title: Ionizing Radiation-induced Proteomic Oxidation in Escherichia coli
Abstract: Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli, the most significant target of IR by a wide margin was glyceraldehyde 3′-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life. Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli, identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli, the most significant target of IR by a wide margin was glyceraldehyde 3′-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life. Ionizing radiation (IR) is a ubiquitous source of lethal cellular damage to all organisms. The main source of such damage is reactive oxygen species (ROS) generated from IR-induced intracellular water radiolysis (1Reisz J.A. Bansal N. Qian J. Zhao W. Furdui C.M. Effects of ionizing radiation on biological molecules–mechanisms of damage and emerging methods of detection.Antioxid. Redox Signal. 2014; 21: 260-292Crossref PubMed Scopus (386) Google Scholar, 2Desouky O. Ding N. Zhou G. Targeted and non-targeted effects of ionizing radiation.J. Rad. Res. App. Sci. 2015; 8: 247-254Crossref Google Scholar, 3Cox M.M. Battista J.R. Deinococcus radiodurans - The consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (518) Google Scholar, 4Maxwell C.A. Fleisch M.C. 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Identification of a new methionine sulfoxide reductase.J. Biol. Chem. 2001; 276: 48915-48920Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and is incapable of repairing damage to the other 18 amino acids. Thus, although a significant number of proteins have evolved to repair DNA damage, there do not appear to be ROS degrading enzymes and protein repair machinery yet evolved which address oxidative damage to the proteome caused by the large scale generation of highly reactive and short lived (nanoseconds) hydroxyl radicals generated by IR (16Cadet J. Wagner J.R. DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation.Cold Spring Harb. Perspect. Biol. 2013; 5 (a012559): a012559Crossref PubMed Scopus (488) Google Scholar). The terrestrial background dose of IR (∼ 2.4 mGy/per year) is not sufficient to cause lethal damage. Thus, IR cannot act as a selective pressure in most organisms (1Reisz J.A. Bansal N. Qian J. Zhao W. Furdui C.M. Effects of ionizing radiation on biological molecules–mechanisms of damage and emerging methods of detection.Antioxid. Redox Signal. 2014; 21: 260-292Crossref PubMed Scopus (386) Google Scholar, 3Cox M.M. Battista J.R. Deinococcus radiodurans - The consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (518) Google Scholar). Indeed, the extremophile levels of IR resistance found in all domains of life has co-evolved with desiccation resistance (3Cox M.M. Battista J.R. Deinococcus radiodurans - The consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (518) Google Scholar, 6Daly M.J. Death by protein damage in irradiated cells.DNA Repair. 2012; 11: 12-21Crossref PubMed Scopus (179) Google Scholar, 17Mattimore V. Battista J.R. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.J. Bacteriol. 1996; 178: 633-637Crossref PubMed Google Scholar). The bacterium Deinococcus radiodurans is the best-studied naturally IR-resistant species. D. radiodurans can survive IR doses more than 5000 Gy, orders of magnitude more than the lethal human dose (3Cox M.M. Battista J.R. Deinococcus radiodurans - The consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (518) Google Scholar, 5Daly M.J. A new perspective on radiation resistance based on Deinococcus radiodurans.Nat. Rev. Microbiol. 2009; 7: 237-245Crossref PubMed Scopus (335) Google Scholar, 18Daly M.J. Gaidamakova E.K. Matrosova V.Y. Vasilenko A. Zhai M. Venkateswaran A. Hess M. Omelchenko M.V. Kostandarithes H.M. Makarova K.S. Wackett L.P. Fredrickson J.K. Ghosal D. Accumulation of Mn(II) in, Deinococcus radiodurans facilitates gamma-radiation resistance.Science. 2004; 306: 1025-1028Crossref PubMed Scopus (484) Google Scholar, 19Tanaka M. Earl A.M. Howell H.A. Park M.-J. Eisen J.A. Peterson S.N. Battista J.R. Analysis of Deinococcus radiodurans's transcriptional response to ionizing radiation and desiccation reveals novel proteins that contribute to extreme radioresistance.Genetics. 2004; 168: 21-33Crossref PubMed Scopus (225) Google Scholar). This is likely because of the presence of both widely conserved and unique DNA repair enzymes and to the extraordinary ROS scavenging capacity of the D. radiodurans metabolome (3Cox M.M. Battista J.R. Deinococcus radiodurans - The consummate survivor.Nat. Rev. Microbiol. 2005; 3: 882-892Crossref PubMed Scopus (518) Google Scholar, 5Daly M.J. A new perspective on radiation resistance based on Deinococcus radiodurans.Nat. Rev. Microbiol. 2009; 7: 237-245Crossref PubMed Scopus (335) Google Scholar, 6Daly M.J. Death by protein damage in irradiated cells.DNA Repair. 2012; 11: 12-21Crossref PubMed Scopus (179) Google Scholar, 18Daly M.J. Gaidamakova E.K. Matrosova V.Y. Vasilenko A. Zhai M. Venkateswaran A. Hess M. Omelchenko M.V. Kostandarithes H.M. 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Conservation and diversity of radiation and oxidative stress resistance mechanisms in Deinococcus species.FEMS Microbiol. Rev. 2019; 43: 19-52Crossref PubMed Scopus (87) Google Scholar). Our current understanding of IR-induced damage to cellular macromolecules has been primarily based on in vitro studies, in which nucleotides, amino acids, peptides, or proteins have been exposed to IR in solution and the products characterized via MS (MS) (1Reisz J.A. Bansal N. Qian J. Zhao W. Furdui C.M. Effects of ionizing radiation on biological molecules–mechanisms of damage and emerging methods of detection.Antioxid. Redox Signal. 2014; 21: 260-292Crossref PubMed Scopus (386) Google Scholar, 23Wang D. Kreutzer D.A. Essigmann J.M. Mutagenicity and repair of oxidative DNA damage: insights from studies using defined lesions.Mutat. Res. 1998; 400: 99-115Crossref PubMed Scopus (427) Google Scholar, 24Fuciarelli A.F. Wegher B.J. Gajewski E. Dizdaroglu M. Blakely W.F. Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry.Radiat. Res. 1989; 119: 219-231Crossref PubMed Scopus (95) Google Scholar, 25Xu G. Chance M.R. Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.Chem. Rev. 2007; 107: 3514-3543Crossref PubMed Scopus (539) Google Scholar, 26Maisonneuve E. Ducret A. Khoueiry P. Lignon S. Longhi S. Talla E. Dukan S. Rules governing selective protein carbonylation.PLoS ONE. 2009; 4: e7269Crossref PubMed Scopus (108) Google Scholar, 27Kempner E.S. Effects of high-energy electrons and gamma rays directly on protein molecules.J. Pharm. Sci. 2001; 90: 1637-1646Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Girod M. Enjalbert Q. Brunet C. Antoine R. Lemoine J. Lukac I. Radman M. Krisko A. Dugourd P. 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Quantitative measurement of radiation-induced base products in DNA using gas chromatography-mass spectrometry.Radiat. Res. 1989; 119: 219-231Crossref PubMed Scopus (95) Google Scholar, 25Xu G. Chance M.R. Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.Chem. Rev. 2007; 107: 3514-3543Crossref PubMed Scopus (539) Google Scholar, 26Maisonneuve E. Ducret A. Khoueiry P. Lignon S. Longhi S. Talla E. Dukan S. Rules governing selective protein carbonylation.PLoS ONE. 2009; 4: e7269Crossref PubMed Scopus (108) Google Scholar, 27Kempner E.S. Effects of high-energy electrons and gamma rays directly on protein molecules.J. Pharm. Sci. 2001; 90: 1637-1646Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Girod M. Enjalbert Q. Brunet C. Antoine R. Lemoine J. Lukac I. Radman M. Krisko A. Dugourd P. Structural basis of protein oxidation resistance: a lysozyme study.PLoS ONE. 2014; 9: e101642Crossref PubMed Scopus (8) Google Scholar, 29Minkoff B.B. Bruckbauer S.T. Sabat G. Cox M.M. Sussman M.R. Covalent modification of amino acids and peptides induced by ionizing radiation from an electron beam linear accelerator used in radiotherapy.Radiat. Res. 2019; 191: 447-459Crossref PubMed Scopus (3) Google Scholar, 35Du J. Gebicki J.M. Proteins are major initial cell targets of hydroxyl free radicals.Int. J. Biochem. Cell Biol. 2004; 36: 2334-2343Crossref PubMed Scopus (183) Google Scholar). However, there have been no reports identifying the proteins that are the immediate in vivo targets of IR-mediated oxidation. To identify and quantify IR-dependent oxidative modifications in vivo, we used modern MS methods and the model single-celled organism Escherichia coli. E. coli is an ideal subject for such comprehensive proteomic studies. Previous work has used MS to characterize the make-up of the E. coli proteome across several growth conditions (36Brown C.W. Sridhara V. Boutz D.R. Person M.D. Marcotte E.M. Barrick J.E. Wilke C.O. Large-scale analysis of post-translational modifications in E. coli under glucose-limiting conditions.BMC Genomics. 2017; 18: 301Crossref PubMed Scopus (27) Google Scholar, 37Potel C.M. Lin M.H. Heck A.J.R. Lemeer S. Widespread bacterial protein histidine phosphorylation revealed by mass spectrometry-based proteomics.Nat. Methods. 2018; 15: 187-190Crossref PubMed Scopus (101) Google Scholar, 38Soufi B. Krug K. Harst A. Macek B. Characterization of the E. coli proteome and its modifications during growth and ethanol stress.Front. Microbiol. 2015; 6: 103Crossref PubMed Scopus (87) Google Scholar, 39Li G.W. Burkhardt D. Gross C. Weissman J.S. 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We quantify the extent of global oxidative modification to the E. coli and D. radiodurans proteomes caused directly by IR treatment and chemistries before any biological response. Unless otherwise stated, Escherichia coli cultures were grown in Luria-Bertani (LB) broth at 37°C with aeration. E. coli were plated on 1.5% LB agar medium and incubated at 37°C. LB broth and agar was prepared as previously described (42Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar). Overnight cultures were grown in a volume of 3 ml for 16 to 18 h. Overnight cultures were routinely diluted 1:100 in 10 ml of LB medium in a 50 ml Erlenmeyer flask and were grown at 37°C with shaking at 200 rpm and were harvested at an OD600 of 0.2. After growth to an OD600 of 0.2, cultures were placed on ice for 10 min to stop growth before being used for assays. Where indicated, E. coli cells were grown in EZ defined rich medium (Teknova; Hollister, CA) supplemented with 0.2% glycerol. Deinococcus radiodurans was grown at 30°C in 2 × TGY broth (1% Tryptone, 0.6% Yeast Extract, 0.2% glucose per 1 L dH20), or plated on 2 × TGY agar (with added 1.5% Bacto Agar per 1 L of media), as previously described (17Mattimore V. Battista J.R. Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.J. Bacteriol. 1996; 178: 633-637Crossref PubMed Google Scholar). Plates were incubated until colonies were easily countable (48–72 h). Overnight cultures were incubated for 24 h before use. To generate exponential phase cultures for biological assays, overnight cultures were diluted 1:100 in 10 ml of 2 × TGY medium in a 50 ml Erlenmeyer flask and were grown at 30 °C with shaking at 200 rpm and were harvested at an OD600 of 0.08–0.15. Exponential phase cultures were placed on ice for 10 min to stop growth before being used for assays. Serial dilations, plating and irradiations were carried out as previously described (43Bruckbauer S.T. Trimarco J.D. Martin J. Bushnell B. Senn K.A. Schackwitz W. Lipzen A. Blow M. Wood E.A. Culberson W.S. Pennacchio C. Cox M.M. Experimental evolution of extreme resistance to ionizing radiation in Escherichia coli after 50 cycles of selection.J. Bacteriol. 2019; 201: e00784-18Crossref PubMed Scopus (15) Google Scholar). One ml sample for each dose tested (including 0 Gy) were removed and aliquoted into sterile 1.5 ml microfuge tubes. Samples were pelleted by centrifugation at 13 × g for 1 min, the supernatant removed, and resuspended in 1 ml ice-cold 1 × PBS (PBS). The pelleting process was repeated three more times to wash cells. A 100 μL aliquot of each culture was removed, serial diluted 1:10 in 900 μL of PBS to a final 10,000-fold dilution and 100 μL was plated on LB agar to determine the colony forming units (CFU)/ml before irradiation. Samples were maintained at 4°C and irradiated with the appropriate doses as described. After irradiation, a 100 μL aliquot of each culture was removed, diluted, and plated to determine CFU/ml and percent survival as described (43Bruckbauer S.T. Trimarco J.D. Martin J. Bushnell B. Senn K.A. Schackwitz W. Lipzen A. Blow M. Wood E.A. Culberson W.S. Pennacchio C. Cox M.M. Experimental evolution of extreme resistance to ionizing radiation in Escherichia coli after 50 cycles of selection.J. Bacteriol. 2019; 201: e00784-18Crossref PubMed Scopus (15) Google Scholar). Biological quintuplicate cultures of E. coli were grown overnight in LB from isolated colonies as described in Growth Conditions. Cultures were then diluted 1:100 in 25 ml LB in 125 ml Erlenmeyer flasks and grown to an OD600 of ∼0.2 as described. Two aliquots of 1 ml of each culture were then washed with 1× PBS and mock-treated or irradiated with 1000 Gy as described in Ionizing Radiation Resistance Assay. Following irradiation, 100 μL of each sample was removed and pelleted via centrifugation at 13 × g for 1 min Supernatants were removed, and pellets were resuspended in 12.5 μL of dH2O and 12.5 μL of 2× Laemilli sample buffer (250 mm Tris-HCl pH 6.8, 10% SDS, 20% glycerol, 10% β-Mercaptoethanol, sufficient Bromphenol Blue for dark blue coloration), boiled 5 min, and placed in 4°C. These samples were then used for the Oxyblot Protein Oxidation Detection Kit (Sigma Aldrich, St. Louis, MO; Cat# S7150) per manufacturer's protocols. A single culture of E. coli was grown overnight in LB from an isolated colony as described in Growth conditions. The culture was then diluted 1:100 in 10 ml LB in 3 separate 50 ml Erlenmeyer flasks and grown to an OD600 of ∼0.2 as described. Four ml of each culture was pelleted by centrifugation at 13 × g for 1 min per ml of culture (removing supernatant in between) and resuspended in 1 ml of 1× PBS. Each aliquot then washed with 1× PBS and irradiated with 10, 100, or 1,000 Gy as described in Ionizing radiation resistance assay. Before irradiation, 100 μμL was removed from each sample to assay CFU/ml of the untreated culture. Samples were lysed by addition SDS to a final concentration of 2.0%, then immediately subjected to protein extraction and concentration using a standard methanol/chloroform protocol. Purified protein pellets were solubilized in 8M urea with 50 mm ammonium bicarbonate (ABC) and subjected to a standard BCA assay to determine protein concentration. Ten micrograms (varying volumes) of each was diluted to 4M urea with 50 mm ABC and treated with 2 mm DTT for 30 min at 50°C, 5 mm iodoacetamide for 30 min at room temperature in darkness, and then 2 mm DTT for 5 min at room temperature. Samples were diluted further to 1M urea with 50 mm ABC, and 0.05 μg of Trypsin and Lys-C proteases were each added (final protease mass/protein mass of 1:100). Samples were incubated overnight at 37°C, for 15 h total. Digestions were stopped with addition of neat formic acid to 1.0%, subjected to solid phase cleanup using Agilent C18 OMIX tips (Agilent Technologies; Santa Clara, CA), according to manufacturer's protocol, and dried down to completion using a vacuum centrifuge. Samples were resolubilized into 0.1% formic acid and injected independently onto an Orbitrap Elite mass spectrometer (Thermo Fisher; Waltham, MA). For liquid chromatographic conditions, stationary phase was C18 and flow rate was 300nL/min. Mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. For separation and elution, a 150-min gradient to 20% buffer B was used followed by a 12 min gradient to 50% B and 5 min gradient to 95%B. Data-dependent acquisition was performed using a top-20 method with MS1 scans occurring at 120K resolving power in the Orbitrap and MS2 fragment ion scans occurring in the ion trap following CID fragmentation with a normalized collision energy of 35.0 units for +2 and greater charge states. Dynamic exclusion was enabled with a repeat count of 1 within a 30 s window. Data were analyzed using the Sequest algorithm within Proteome Discoverer (PD) (Thermo Fisher). The Uniprot K12 E. coli proteome, downloaded on 7/2/2019 (PID: UP000000625, 4382 sequences including contaminants), was searched with the specified parameters: trypsin with 2 possible missed cleavages, precursor and fragment mass tolerance 10 ppm and 0.6 Da, respectively, and a max amount of 4 dynamic modifications per peptide. Dynamic modifications were specified as carbamidomethyl/+57.021 Da (on C), oxidation/+15.995 Da (on CDEFHILMNPQRSTVWY), carbonylation/+13.979 Da (on AEILQRSV), dioxidation/+31.990 Da (on CEFILMPRVWY), and trioxidation/+47.985 Da (on CFWY). Searches were based on previous reports of abundance of the given modifications on each amino acid reside (25Xu G. Chance M.R. Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.Chem. Rev. 2007; 107: 3514-3543Crossref PubMed Scopus (539) Google Scholar). A false discovery rate (FDR) for peptide spectral matches (PSMs), peptides, and proteins of 0.05% was used via percolator in PD. E. coli or D. radiodurans cultures were grown overnight in biological quintuplicate and to an OD600 of 0.2 or 0.1 in EZ + 0.2% glycerol or 2× TGY, respectively in a total volume of 50 ml for each replicate. A 40 ml aliquot of each early exponential phase culture was pelleted by centrifugation at 3500 rpm for 10 min at 4°C. Supernatants were poured off, and samples resuspended in 40 ml of ice-cold 1× PBS. This process was repeated twice more with cells resuspended in 20 ml ice-cold PBS, and a final time suspending in 500 μL ice-cold 1× PBS. Four 100 μL aliquots were made for each culture in 1.5 ml microfuge tubes. Two for 0 and 1000 Gy for MS analysis, and two for plating to determine lethality. A cartoon illustration of the protocol is depicted in supplemental Fig. S2. Samples were lysed by addition SDS to a final concentration of 2.0%, then immediately subjected to protein extraction and concentration using a standard methanol/chloroform protocol. Purified protein pellets were solubilized in 8M urea with 50 mm triethylammonium bicarbonate (TEAB) and subjected to a standard BCA assay to determine protein concentration. For each of the 10 samples, 10 μg (varying volumes) of each was diluted to 4M u