Nitration of Tyrosine 92 Mediates the Activation of Rat Microsomal Glutathione S-Transferase by Peroxynitrite

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an ∼2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue. There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an ∼2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue. The microsomal glutathione S-transferase 1 (MGST1) 2The abbreviations used are: MGST1, microsomal glutathione S-transferase 1; ONOO–, peroxynitrite; RNS, reactive nitrogen species; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; ESI-MS/MS, electrospray ionization mass spectrometry; LLC-PK1 cells, porcine kidney epithelial cells; DTPA, diethylenetriaminepentaacetic acid; ACN, acetonitrile; GSNO, S-nitrosoglutathione; aa, amino acids. is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism superfamily of proteins (1Jakobsson P.J. Morgenstern R. Mancini J. Ford-Hutchinson A. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (302) Google Scholar). Although these proteins share common structural characteristics, their biological functions are quite varied, including roles in xenobiotic metabolism, cellular protection, pain, and inflammation. Human membrane-associated proteins in eicosanoid and glutathione metabolism includes six proteins: 5-lipooxygenase-activating protein, leukotriene-C4 synthase, MGST1, MGST2, MGST3, and microsomal prostaglandin-E synthase. Rat hepatic MGST1 exists as a homotrimer of identical 17.3-kDa subunits and has a high degree of amino acid sequence similarity (83%) to human MGST1. In contrast to the cytosolic GSTs, an important characteristic of microsomal GST1 is that the enzyme can be activated by various treatments, including limited proteolysis, heating, radiation, and exposure to sulfhydryl modifying reagents and reactive oxygen species. In addition, since MGST1 possesses glutathione peroxidase activity, it has been suggested that this enzyme may play a protective role under conditions of oxidative stress (2Mosialou E. Morgenstern R. Arch. Biochem. Biophys. 1989; 275: 289-294Crossref PubMed Scopus (70) Google Scholar, 3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). Activation of MGST1 also occurs after exposure to reactive nitrogen species (RNS), including nitric oxide (NO) donors and peroxynitrite (ONOO–) (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar, 4Ji Y. Toader V. Bennett B.M. Biochem. Pharmacol. 2002; 63: 1397-1404Crossref PubMed Scopus (46) Google Scholar), in contrast to the cytosolic GSTs, which are inhibited by RNS (5Wong P.S.-Y. Eiserich J.P. Reddy S. Lopez C.L. Cross C.E. Vliet A.V.D. Arch. Biochem. Biophys. 2001; 394: 216-228Crossref PubMed Scopus (75) Google Scholar). The ONOO–-induced activation of MGST1 was associated with tyrosine nitration, polymer formation, and protein fragmentation but not protein S-oxidation (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). This increase in enzyme activity by ONOO– is unusual, since tyrosine nitration of proteins is almost always associated with a loss of function. For example, a number of enzymes involved in protection against oxidative stress are inhibited by ONOO–, including superoxide dismutases (6Ischiropoulos H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1429) Google Scholar), glutathione reductase (7Francescutti D. Baldwin J. Lee L. Mutus B. Protein Eng. 1996; 9: 189-194Crossref PubMed Scopus (48) Google Scholar), cytosolic GSTs (5Wong P.S.-Y. Eiserich J.P. Reddy S. Lopez C.L. Cross C.E. Vliet A.V.D. Arch. Biochem. Biophys. 2001; 394: 216-228Crossref PubMed Scopus (75) Google Scholar), catalase (8Grzelak A. Soszynski M. Bartosz G. Scand. J. Clin. Lab. Invest. 2000; 60: 253-258Crossref PubMed Scopus (31) Google Scholar, 9Kocis J.M. Kuo W.N. Liu Y. Guruvadoo L.K. Langat J.L. Front. Biosci. 2002; 7: 175-180Crossref Scopus (15) Google Scholar), and glutathione peroxidase (8Grzelak A. Soszynski M. Bartosz G. Scand. J. Clin. Lab. Invest. 2000; 60: 253-258Crossref PubMed Scopus (31) Google Scholar, 10Padmaja S. Squadrito G.L. Pryor W.A. Arch Biochem. Biophys. 1998; 349: 1-6Crossref PubMed Scopus (108) Google Scholar). The activation of MGST1 by RNS, together with its GSH peroxidase activity, suggest that this enzyme may play an important role in limiting the extent of oxidative tissue injury under pathophysiological conditions of excessive ONOO– formation, especially if other antioxidant defense mechanisms are compromised. The tyrosine residues of MGST1 that are nitrated by ONOO– have not been identified. In the present study, we used two mass spectrometric techniques, MALDI-TOF MS and ESI-MS/MS, to identify the tyrosine residues of purified rat hepatic MGST1 that are nitrated after ONOO– treatment. Subsequently, we determined the functional consequences resulting from nitration of these tyrosine residues using site-directed mutagenesis. Materials—Hydroxyapatite, CM-Sepharose, GSH, 1-chloro-2,4-dinitrobenzene, Triton X-100, manganese (IV) dioxide, Dulbecco's modified Eagle's medium, and nutrient mixture F-12 were purchased from Sigma. Sequence-grade modified trypsin was from Promega (Madison, WI). Monoclonal anti-nitrotyrosine antibody was from Cayman Chemical Co. (Ann Arbor, MI) and horseradish peroxidase-linked goat anti-mouse IgG was obtained from Bio-Rad (Mississauga, Ontario, Canada). Chemiluminescence reagents were from Kirkegaard and Perry (Gaithersburg, MA). All other chemicals were of reagent grade and were obtained from common commercial sources. Enzyme Purification and Enzyme Activity Assay—Rat hepatic MGST1 was purified from male Sprague-Dawley rats (250–300 g) by hydroxyapatite and CM-Sepharose chromatography as described (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). The purity of the enzyme preparation was assessed by SDS-PAGE on 15% gels under reducing conditions. The protein migrated as a single band at about 17 kDa (4Ji Y. Toader V. Bennett B.M. Biochem. Pharmacol. 2002; 63: 1397-1404Crossref PubMed Scopus (46) Google Scholar). Prior to experiments, GSH was removed from the preparation by dialysis against buffer containing 10 mm potassium phosphate, pH 7.0, 0.1 mm EDTA, 1% Triton X-100, and 20% glycerol using a System 500 Microdialyzer (Pierce). Enzyme activity was determined by the spectrophotometric method of Habig et al. (11Habig W.H. Pabst M.J. Jacoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). Samples (1.0 ml) contained 100 mm potassium phosphate, pH 6.5, 0.5% Triton X-100, 1.0 mm GSH, and 1.0 mm 1-chloro-2,4-dinitrobenzene at 25 °C. Preparation of Peroxynitrite (ONOO–) and Treatment of MGST1—ONOO– was synthesized from acidified nitrite and H2O2 as described (12Beckman J.S. Chen J. Ischiropoulos H. Crow J.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (970) Google Scholar). Prior to each experiment, excess H2O2 in ONOO– solutions was removed by MnO2 chromatography (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). Purified enzyme (20 μg/ml) or microsomal fractions (200–400 μg/ml protein) in 100 mm potassium phosphate, pH 7.0, containing 100 μm diethylenetriaminepentaacetic acid (DTPA), were exposed to the indicated concentrations of ONOO– at room temperature for 10 s. To keep the pH value of the reaction unchanged, ONOO– was added to the protein or microsomal solution as a small volume during vigorous mixing, and the reaction was terminated by dilution of the sample into the assay buffer for the determination of enzyme activity or into the sample buffer for SDS-PAGE. For immunoblot analysis, samples treated with ONOO– were resolved on a 15% SDS-PAGE gel under non-reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes and incubated with a 3-nitrotyrosine-specific antibody. The immunoreactive protein bands were visualized by enhanced chemiluminescence. Identification of Nitration Sites by MALDI-TOF MS—Protein bands corresponding to the MGST1 monomer (17.3 kDa) with and without ONOO– treatment were excised from Coomassie Blue-stained, 15% reducing SDS-PAGE gels (approximately 2 μg of purified protein/band). Gel slices were washed three times with 50% ACN, 25 mm ammonium bicarbonate, pH 8.0 (15 min each time under gentle agitation) to remove the Coomassie Blue and were then incubated with 100% ACN for 5 min and vacuum-dried. To reduce and alkylate proteins, dried gel slices were incubated with 10 mm DTT in 100 mm ammonium bicarbonate at 55 °C for 45 min followed by incubation with 55 mm iodoacetamide in 100 mm ammonium bicarbonate for 30 min at room temperature in the dark. Gel slices were then washed with 25 mm ammonium bicarbonate and dried with 100% ACN as described above. In-gel digestion was performed by swelling the dried gel slices with 10 μg/ml trypsin in 25 mm ammonium bicarbonate, pH 8.0, for 30 min on ice. Excess trypsin solution was removed and digestion continued at 37 °C for 16 h. Tryptic peptides were extracted with 50% ACN/5% trifluoroacetic acid, dried under vacuum, and reconstituted in 3 μl of 70% ACN, 0.1% trifluoroacetic acid. Reconstituted extract (0.5 μl) was mixed with 0.5 μl of matrix (10 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid in 70% ACN, 0.1% trifluoroacetic acid), spotted onto a stainless steel 100-well MS plate, and air dried. Samples were analyzed using a Voyager DE-Pro MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) operated in the delayed extraction/reflector mode with an accelerating voltage of 20 kV, grid voltage setting of 72%, and a 50-ns delay. Three spectra (100 laser shots/spectrum) were obtained for each sample. External calibration was performed using a Sequazyme Peptide Mass Standard kit (Perseptive Biosystems, Framingham, MA) containing the following standards: des-Arg-bradykinin, angiotensin-1, and Glu-fibrinopeptide B. Peptide mass fingerprinting was conducted with the data using Protein at The to nitration were in the for and The for of the protein by MALDI-TOF MS a of at peptides from the protein with the data of rat proteins with to mass and with an of than for including modified of by were analyzed with a of to a mass spectrometer CA) with a ionization was performed using a MA) with a μm and a μm The was 5 μm matrix in a of 5 The was ACN, 0.1% trifluoroacetic acid. of peptides was using a of ACN from 5 to in 45 followed by a with spectra were analyzed using For the a 2 was for peptides with a of 2 to the whereas the sequence of peptides with a was containing rat MGST1 was a from Morgenstern of The was with and the MGST1 was into All were using the site-directed mutagenesis kit was used as for mutants Y92F and Y153F, and was used as the for of double mutants and at the residues to be were synthesized by Ontario, as Y92F and Y153F All were by and cells were obtained from the and in Dulbecco's modified Eagle's with 5 μg/ml 2 mm 10 mm pH and μg/ml For LLC-PK1 cells were in and the were using to the incubation with the cells were by in 100 mm potassium phosphate, pH and microsomal fractions by MGST1 activity in and microsomal fractions was assessed as described above. MGST1 activity were performed in and data are as the were analyzed by the as were Nitration and Activation of MGST1 by of purified MGST1 to mm ONOO– resulted in a increase in enzyme activity ONOO– or mm H2O2 not enzyme activity The of in the activated protein was demonstrated using SDS-PAGE non-reducing and immunoblot a anti-nitrotyrosine antibody. of the 17.3-kDa MGST1 monomer in after exposure to increasing of ONOO– In addition, there was in proteins bands of about and as (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar), the of and of purified MGST1 by Purified MGST1 (20 μg/ml) was exposed to the indicated concentrations of ONOO– in 100 mm potassium phosphate, pH 7.0, containing 100 μm DTPA, at room temperature for 10 s. Nitration of MGST1 was by SDS-PAGE and immunoblot using antibody There were three bands with and corresponding to the MGST1 and The was and with to of MGST1 Identification of Sites of Nitration in and MGST1 were by 15% and the MGST1 (17.3 kDa) digestion with Peptide mass fingerprinting by MALDI-TOF MS the of the 17.3-kDa band as rat MGST1 number protein for and There are tyrosine residues in MGST1 Tryptic peptides containing these tyrosine residues were by MALDI-TOF MS with the of the containing this was not to ionization of the containing on the sequence of MGST1, digestion using or not have been to peptides containing with a number of peptides not for by MALDI-TOF of mass spectrometric of MS and and in a The containing was in and MGST1 by ESI-MS/MS, whereas the nitrated of the was only in the sample. and present the were using of but there was evidence for nitration of of these tyrosine residues in and the residues Nitration of these two tyrosine residues also was not in the residue at was always in the and this could have the of other the The tyrosine residue the was in a acid by or as a that resulted from a single the 5 amino by MALDI-TOF Nitration at in samples was in The MS data are in The for and nitrated peptides in samples and a of the sequence for peptides containing nitrated are in functional consequences of nitration of tyrosine residues and of MGST1 were determined by the wild type protein to MGST1 in which these tyrosine residues were with or in (Y92F, Y153F, and a Y92F, Y153F double These nitration at the amino acid site containing the wild type or MGST1 single and double were in LLC-PK1 cells and the MGST1 activity in the microsomal fractions MGST1 protein was in cells but not in cells or cells with with a increase in MGST1 activity in cells. the MGST1 activity was in containing the wild type or after treatment with mm ONOO– for 10 there was a increase in MGST1 activity only in microsomal fractions from cells with wild type MGST1 or with the Y153F mutant In MGST1 activity in the Y92F mutant or the double mutant was by ONOO–, that nitration of is for the activation of MGST1. MGST1 is a protein, with each containing seven tyrosine residues positions and in to a single residue of the seven tyrosine residues are in the of the of of MGST1 has been used to a of the In this the enzyme three of a P.J. Morgenstern R. H. Biophys. 2002; PubMed Scopus (46) Google Scholar). The of the enzyme is to be contained in the cytosolic of the protein and A. two of the tyrosine residues and are in the in the cytosolic and the at the the other are in one of the P.J. Morgenstern R. H. Biophys. 2002; PubMed Scopus (46) Google Scholar). The cytosolic not tyrosine residues P.J. Morgenstern R. H. Biophys. 2002; PubMed Scopus (46) Google but the or oxidative of which results in activity and is to as a of or oxidative stress P.J. H. Morgenstern R. PubMed Scopus Google Scholar). of by reactive oxygen species not to enzyme since we 3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google and and G. Morgenstern R. Biochem. Pharmacol. 1992; PubMed Scopus Google could not activation of the enzyme by H2O2 or by superoxide In that have demonstrated activation of the enzyme by H2O2 Y. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, Y. Arch. Biochem. Biophys. 1992; PubMed Scopus Google Scholar), with H2O2 were performed in the of GSH, and the increase in enzyme activity was attributed to or polymer formation, rather than of the In with H2O2 were performed in the of GSH, and of and activation of the enzyme not be To identify of tyrosine nitration of MGST1, we mass spectrometric using and to of the protein and of of the tyrosine of the of the MGST1 obtained using the of nitration of in but not in the nitration site at was by in of the other tyrosine and was not was not by MS and we could not its nitration is that the residue present in the that and was always in the was not present in peptides containing or of is a common that occurs to protein preparation as as during protein and can of ionization and can or of other the of the or of nitration at have to be by other by of the to its ionization or by other as with site specific Peroxynitrite is a biological oxidant that has been in of tissue can with a of biological to cellular and and there are reports in the describing the of ONOO– on the activity of a of proteins, including enzymes involved in defense against oxidative In this activity has been attributed to nitration of tyrosine in of sulfhydryl J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1999; PubMed Google Scholar, Beckman J.S. Crow J.P. Arch. Biochem. Biophys. 1999; PubMed Scopus Google or of site J.P. Beckman J.S. J.M. PubMed Scopus Google Scholar, J. Clin. Invest. 2002; PubMed Scopus Google has been In treatment with ONOO– MGST1 activity in hepatic or in purified enzyme 3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google and and also the GSH peroxidase activity of the enzyme (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). In a study, we that MGST1 was to sulfhydryl than to tyrosine nitration (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). of MGST1 to 0.1 mm ONOO– resulted in of sulfhydryl whereas tyrosine nitration was the extent of by ONOO– not with the increase in enzyme activity, that S-oxidation of MGST1 by ONOO– was not to the enzyme In the increase in tyrosine nitration by ONOO– quite the increase in enzyme activity, that tyrosine nitration was the for the gain of function (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). The results of the site-directed mutagenesis evidence that this is the since in cells expressing the Y92F mutant, ONOO– was without The site of MGST1 is on the cytosolic of R. G. Morgenstern R. Biophys. 1994; PubMed Scopus Google Scholar). The MGST1 one of GSH, and is to play a role in the activation of the enzyme Morgenstern R. Biochem. J. PubMed Scopus Google Scholar, R. R. S. Morgenstern R. 2000; PubMed Scopus Google Scholar). a with of GSH followed by of R. J. Morgenstern R. PubMed Scopus Google Scholar). The about by of by the of and Although nitration of was to this residue is the removed from the site of the and not be to enzyme or This is by the site-directed mutagenesis data in which from cells expressing the Y153F mutant in MGST1 activity after exposure to the other is in the on the of the cytosolic containing the the of it to that in a to that after structural of a about by nitration of this tyrosine residue increase the of and increase in with the role of as a of and oxidative the nitration of could function as a of from this and other have that activation of MGST1 by RNS occurs by two of and nitration of and the as to the of these two mechanisms in the purified the degree of activation by ONOO– is about than that by (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar, 4Ji Y. Toader V. Bennett B.M. Biochem. Pharmacol. 2002; 63: 1397-1404Crossref PubMed Scopus (46) Google Scholar). hepatic enzyme activity is about by ONOO– and is not by in purified enzyme or microsomal ONOO– is a of the The of of could be to of the microsomal by cytosolic GSTs, activity is inhibited by GSNO, with a of in in a in which of MGST1 was assessed using evidence was for of the enzyme after incubation of hepatic with or in from rats treated with to stress Pharmacol. PubMed Scopus Google Scholar). that in under associated with ONOO– formation, that tyrosine nitration be the of MGST1. In a we that of MGST1 to microsomal a in with the that the GSH peroxidase activity of MGST1 is after treatment with ONOO– and a protective function for the nitrated enzyme (3Ji Y. Bennett B.M. Mol. Pharmacol. 2003; 63: 136-146Crossref PubMed Scopus (40) Google Scholar). conditions of antioxidant enzymes are inactivated by S-oxidation tyrosine nitration by ONOO– (5Wong P.S.-Y. Eiserich J.P. Reddy S. Lopez C.L. Cross C.E. Vliet A.V.D. Arch. Biochem. Biophys. 2001; 394: 216-228Crossref PubMed Scopus (75) Google Scholar, H. Zhu L. Chen J. Tasi M. Martin J.C. Smith C.D. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 431-437Crossref PubMed Scopus (1429) Google Scholar, D. Baldwin J. Lee L. Mutus B. Protein Eng. 1996; 9: 189-194Crossref PubMed Scopus (48) Google Scholar, A. Soszynski M. Bartosz G. Scand. J. Clin. Lab. Invest. 2000; 60: 253-258Crossref PubMed Scopus (31) Google Scholar, 9Kocis J.M. Kuo W.N. Liu Y. Guruvadoo L.K. Langat J.L. Front. Biosci. 2002; 7: 175-180Crossref Scopus (15) Google Scholar, 10Padmaja S. Squadrito G.L. Pryor W.A. Arch Biochem. Biophys. 1998; 349: 1-6Crossref PubMed Scopus (108) Google Scholar). The data obtained in the present suggest that nitration of and activation of MGST1 after exposure to ONOO– may be an important for cellular protection against stress under in which other antioxidant defense mechanisms are compromised. of of and the Protein at for in the MALDI-TOF and

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