- Email: [email protected]
mass spectrometry assay for 3-nitrotyrosine
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[351 Gas Chromatography/Mass Spectrometry Assay for 3-Nitrotyrosine By MICHAELBALAZY
Introduction Measurements of 3-nitrotyrosine levels in biological samples are being used to evaluate exposure to reactive nitrogen species in vivo. Because protein tyrosine nitration has been detected in many pathophysiological conditions, 1-3 it is thought that potent nitrating compounds are generated during disease processes. A product of nitric oxide (NO) and superoxide, the peroxynitrite (ONOO-) has been shown originally to cause tyrosine nitration.4 Recent work also suggests that other nitrating molecules, such as nitryl chloride and nitrogen dioxide, may be generated in vivo from the oxidation of nitrite by peroxidases. 5'6 Measurement of 3-nitrotyrosine in animal studies, as well as studies involving human subjects, can lead to a better understanding of the basic biochemical principles involved in free radical damage mediated by reactive nitrogen species. Furthermore, pharmacological control of the synthesis of 3-nitrotyrosine-labeled proteins might lead to alleviation of many of the symptoms associated with such damage. One of the factors that inhibit progress in this area is the difficulty of quantifying 3-nitrotyrosine in proteins, as sensitive methods are required to detect small quantities of 3-nitrotyrosine. Methods currently available for the measurement of 3-nitrotyrosine based on competitive binding with immunoglobulins raised against the 3-nitrotyrosine- or 3-nitrotyrosine-containing peptides and on HPLC suffer from a lack of sensitivity and/or specificity. Various immunoassay techniques, including enzyme-linked immunoassay, have been reported to measure 3-nitrotyrosine-containing proteins. 7,8 These assays represent very sensitive means by which one can detect these particular products. Although the antibodies are useful to localize 3-nitrotyrosine in tissues, it is difficult to quantify 3-nitrotyrosine using these techniques. 9'1° Several 1 K. A. Hanafy, J. S. Krumenacker, and E Murad, Med. Sci. Monit. 7, 801 (2001). 2 S. A. Greenacre and H. Ischiropoulos, Free Radic. Res. 3d, 541 (2001). 3 C. Oldreive and C. Rice-Evans, Free Radic. Res. 35, 215 (2001). 4 H. Ischiropoulos, L. Zhu, J. Chen, M. Tsai, J. C. Martin, C. D. Smith, and J. S. Beckman, Arch. Biochem. Biophys. 298, 431 (1992). 5 j. p. Eiserich, M. I-Iristova, C. E. Cross, and A. D. Jones, et al., Nature 391, 393 (1998). 6 L. Gebicka, Acta Biochim. Pol. 46, 919 (1999). 7 j. Khan, D. M. Brennan, N. Bradley, and B. Gao, et al., Biochem. J. 330, 795 (1998). s y. Z. Ye, M. Strong, Z. Q. Huang, and J. S. Beckman, Methods Enzymol. 269, 201 (1996). 9 A. van der Vliet, J. P. Eiserich, H. Kaur, and C. E, Cross, et al., Methods EnzymoL 269, 175 (1996). l0 j. p. Crow and H. Ischiropoulos, Methods Enzymol. 269, 185.
METHODSIN ENZYMOLOGY,VOL.359
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HPLC assays have been reported that can separate and detect 3-nitrotyrosine with ultraviolet or electrochemical detectors, 1°A1 and detection limits in the range of several micrograms per milligram of protein have been reported. 1° Reduction of 3-nitrotyrosine with dithionite generates 3-aminotyrosine, which can be detected using fluorescence or electrochemical detectors, and enhances the sensitivity 10- to 50-fold relative to the ultraviolet detection. 1°'12 A major disadvantage of the assays based on HPLC is the lack of specificity because artifacts frequently interfere with 3-nitrotyrosine peaks.13 Mass spectrometry is, in principle, an ideal method for detection and quantification of 3-nitrotyrosine because of its sensitivity, specificity, and selectivity. 14 Methods that have been developed mostly rely on converting 3-nitrotyrosine into a more volatile molecule by a series of reactions called derivatization. In this process the carboxyl, hydroxy, and amino groups are converted into an ester, ether, and amide. The final derivative should produce a sharp chromatographic peak and a useful ion for quantification. In an ideal case, a 3-nitrotyrosine derivative that generates a single ion without other fragments will provide the highest sensitivity because it will be converted quantitatively into a detectable ion. This task is difficult to achieve because known 3-nitrotyrosine derivatives produce significant fragmentation largely because they employ several reactions with different chemicals. 14-17 While heptafluorobutyryl and pentafluoropropionyl derivatives of amino acids have been synthesized TM and used for 3-nitrotyrosine quantification, 17,19 it appears that noncharacteristic ions such as C2F5COO-, [C2F5COO2H - F]-, and C2FaCOOare more abundant than ions specific for the amino acid structure.18 In addition, the electron capture mass spectra of these derivatives depend strongly on the amount of sample entering the ion source and, therefore, show considerable variation across the width of the gas chromatographic peak. 18 This could lead to problems in quantitative analysis when working with pentafluoropropionate derivatives. 18 Use of these reagents poses an additional complication because the 3-nitrotyrosine hydroxyl group needs to be further protected with another reagent. 11K. Hensley,M. L. Maidt, Q. N. Pye, C. A. Stewart, M. Wack,T. Tabatabaie, and R. A. Floyd,Anal. Biochem. 251, 187 (1997). 12M. K. Shigenaga,H. H. Lee, B. C. Blount, S. Christen, E. T. Shigeno,H. Yip, and B. N. Ames,Proc. Natl. Acad. Sci. U.S.A. 94, 3211 (1997). t3 H. Kaur, L. Lyras, P. Jenner, and B. Halliwell, J. Neurochem. 70, 2220 (1998). 14M. T. Frost, B. Halliwell, and K. P. Moore, Biochem. J. 345(Pt 3), 453 (2000). t5 E. Schwedhelm,D. Tsikas, E M. Gutzld, and J. C. Frolich,Anal. Biochem. 276, 195 (1999). 16S. Pennathur, V. Jackson-Lewis, S. Przedborski, and J. W. Heinecke, J. Biol. Chem. 274, 34621 (1999). 17j. R. Crowley,K. Yarasheski, C. Leeuwenburgh,J. Turk, and J. W. Heinecker,Anal. Biochem. 259, 127 (1998). 18G. K. C. Low and A. M. Duftield, Biomed. Mass Spectrom. 11, 223 (1984). 19C. Leeuwenburgh,M. M. Hardy, S. L. Hazen, P. Wagner, S. Oh-ishi, U. P. Steinbrecher, and J. W. Heinecke, Z Biol. Chem. 272, 1433 (1997).
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PEROXYNITRITE
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Pentafluorobenzyl (PFB) esters are unique in that they can generate abundant carboxylate anions in negative ion chemical ionization mass spectrometry for compounds such as acidic lipids. 2° We found that 3-nitrotyrosine can be convened into a novel PFB derivative in a single step, and this derivative produces a single ion suitable for a sensitive and quantitative GC/MS assay.21 Chemicals Pentafluorobenzyl bromide (a-bromo-2,3,4,5,6-pentafluorotoluene), diisopropylethylamine, nitronium borofluorate (NO2BF4), and 3-nitro-Ltyrosine are purchased from Aldrich Chemical Co, (Milwaukee, WI) L-4-Hydroxyphenyl-[13C6 ]alanine ([13C6]tyrOsine,isOtOpic abundance >99%; [12C]tyrosine <0.01%) is from Isotec Inc. (Miamisburg, OH) [3,5-3H2]tyrosine (specific activity 59.7 Ci/mmol) is from New England Nuclear (Cambridge, MA) Peroxynitrite has been synthesized in a quenched-flow reactor as described previously22 All solvents are of chromatographic purity from Fisher Scientific. P r e p a r a t i o n of 13C-Labeled 3 - N i t r o t y r o s i n e a s a n I n t e r n a l S t a n d a r d for M a s s S p e c t r o m e t r y One hundred micrograms of [13C6]tyrosine and l0/zCi of 3H-labeled tyrosine (as separate solutions in water/ethanol, 98 : 2) are placed in a dry glass tube, and the solvent is evaporated under vacuum to dryness. The residue is dissolved in 100/zl of a l M solution of NO2BF4 in 0.I M HCI with vigorous vortexing. The NO2BF4 solution should be prepared shortly before use. The tube is then capped with a Teflon cap and placed in a Pierce laboratory heater, with temperature adjusted to 70 °, for 60 min with occasional shaking. After the reaction mixture is cooled, aliquots (20 #l) are injected into a C18 column (150 x 4.6 mm, Beckman, Fullerton, CA) installed in a HPI050 HPLC system (Hewlett-Packard, Palo-Alto, CA). Samples are eluted with a gradient of acetonitrile in water (0 to 100% in 20 rain) at a flow rate of 1 ml/min. The reaction products can be detected by a radioactivity monitor (Packard, Meriden, CT) with an Ecolite (ICN, Costa Mesa, CA) scintillation cocktail and/or a UV detector (205 nm). The effluent from the column is collected into glass tubes as 1-ml fractions using a Gilson FC203B fraction collector. The radiolabeld products in these fractions are identified by scintillation counting of 50-/zl aliquots. [13C6]-3-Nitrotyrosine is collected in a single fraction elufing at 5.5-6.5 rain. The final stock solution is obtained in 5 ml 20 M. Balazy, J. Biol. Chem. 266, 23561 (1991). 21 H. Jiang and M. Balazy, Nitric Oxide 2, 350 (1998). 22 M. Balazy, Pol. J. Pharmacol. 46, 593 (1994).
[35]
GC/MS oF 3-NITROTYROSINE
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of 0.1 M HCI. The concentration of this stock solution is finally determined via scintillation counting of radioactivity in a 100-/xl aliquot. Finally, a solution having a concentration of 0.1 ng [13Cr]-3-nitrotyrosine/ml is prepared. Our attempts to synthesize [13C6]-3-nitrotyrosine using tetranitromethane23 have not been successful. We experienced low yields and difficulty removing a major closely eluting contaminant by HPLC chromatography. Treatment of tyrosine with NO2BF4 gives typical yields of 3-nitrotyrosine in the range of 80-90%. A minor product, 3-nitrohydroxyphenyllactic acid (10-20%), is well separated from 3-nitrotyrosine (Fig. 1). Thus the reaction of NO2BF4 with tyrosine occurs via electrophilic aromatic nitration, whereas substitution of the amino group by a hydroxyl, possibly via an unstable diazonium salt intermediate, is a minor process. Because nitration occurs at carbons labeled with tritium, this reaction also generates a tritium-labeled hydroborotetrafiuoric acid by-product, which elutes as an early peak of radioactivity (Fig. 1). In our experience, use of the radioactive tracer, [3,5-3H2]tyrosine, is essential to monitor the progress of the reaction, as well as in establishing the final concentrations of [13C6]-3-nitrotyrosine solutions. It is also important to note that because one tritium was removed and replaced by an NO2 group, the specific activity of the final product decreases by 50% relative to the specific activity of the original radiolabeled tyrosine. This observation should be taken into account when calculating the final concentration of the [13C6]-3nitrotyrosine stock solution. Analysis by GC/MS of the product eluting at 5.56.5 min confirmed the structure of pure 13C-labeled 3-nitrotyrosine (Fig. 1). P r e p a r a t i o n of P e n t a f l u o r o b e n z y l Derivative of 3 - N i t r o t y r o s i n e The PFB derivative of 3-nitrotyrosine is prepared by the addition of PFB bromide (40/zl) and diisopropylethylamine (40/xl) followed by vigorous mixing and heating at 70 ° for 40 rain. Both reagents for derivatization are prepared as I0% solutions in acetonitrile. Derivatization is terminated by the evaporation of reagents under a genre stream of nitrogen. The residue is then dissolved in 50/zl of methanol and is subjected to purification by HPLC using an acetonitrile gradient in water (50 to I00% in 20 rain; Cl8 column, 150 × 4.6 nun, Beckman). The PFB derivative of 3-nitrotyrosine is typically collected at 13-14 rain. After evaporation of the solvent under vacuum, samples are dissolved in 50/zl of n-decane and analyzed by GC/MS (Fig. 2). The reaction of PFB bromide with 3-nitrotyrosine in the presence of catalytic amounts of diisopropylethylamine quantitatively generates a new derivative (Fig. 3). Treatment of this derivative with a siliconizing compound, BSTFA [N,O-bis(trimethylsilyl)trifluoroacetan~de], does not alter its mass spectrum, indicating that, in addition to the carboxyl, both the hydroxyl and the amino group also reacted with PFB bromide and, therefore, could not be converted into a TMS 23M. Sokolovsky,J. F. Riordan,and B. L. Vallee,Biochemistry5, 3582 (1966).
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(trimethylsilyl) derivative. A hydroxyl vicinal to a nitro group in 3-nitrotyrosine has a pKa of approximately 7.5, which makes it sufficiently acidic to react quantitatively with PFB bromide yielding a PFB ether. This is an uncommon reaction, as primary and secondary alcohols usually do not react with PFB bromide. The heptafluoro derivative of 3-nitrotyrosine requires additional derivatization of the hydroxyl with TBDMS prior to GC/MS analysis. 14 GC/MS Analysis of 3-Nitrotyrosine PFB Ester Analysis can be performed on a suitable GC/MS system equipped with a chemical ionization source and negative ion optics. We have used a HP5989A mass spectrometer interfaced to a HP 5890 gas chromatograph (Hewlett-Packard). Samples (I/zl) are injected with an autoinjector into a capillary chromatographic column (DB-I fused silica, 10 m, 0.25 mm i.d., 0.25 ]zm film thickness, J&W Scientific, Rancho Cordova, CA) via a splitless injector having a temperature of 250 °. The oven temperature is programmed from 150 to 300 ° at 30 °/min during the analysis. Helium is used as the carder gas with a linear velocity of 4 m/sec. Electron-capture ionization is carded out with methane as a moderating gas delivered to produce a pressure in the ion source of 2.8 Torr. The other mass spectrometer parameters are as follows: ion source temperature, 200°; electron energy, 220 eV; and transfer line temperature, 250 ° . Selected ion monitoring is used to record ion abundances at m/z 585 and 591, which correspond to the PFB derivatives of 3-nitrotyrosine and [13C6]-3-nitrotyrosine (internal standard), respectively. Areas under chromatographic peaks defined for ions at m/z 585 and 591 are obtained from GC/MS integration data (Chemstation software). A standard curve is prepared by mixing 3-nitrotyrosine (0 to 40 ng) and [13C6]-3-nitrotyrosine (5 ng) followed by derivatization, purification, and GC/MS analyses as described earlier. The amount of 3-nitrotyrosine in the sample is calculated from the regression line that shows good linearity ( r 2 = 0.999) when plotted as a function of 3-nitrotyrosine concentration, and the ratio of the peak areas is generated by ions m/z 585 and 591 (Fig. 2). A detection limit of 10 pg for the 3-nitrotyrosine PFB derivative has been achieved with a signal-to-noise ratio greater than 3.
[35]
GC/MS OF 3-NITROTYROSINE
397
The tri-PFB derivative of 3-nitrotyrosine shows a sharp and symmetrical chromatographic peak and a simple mass spectrum (Figs. 1 and 2). A prominent ion at m/z 591 corresponds to a carboxylate anion of [13C6]-3-nitrotyrosine and originates from the electron-capture-mediated loss of the PFB radical typically observed in these esters. Formation of such an anion is a favorable process because of its high efficiency and intrinsic stability of the carboxylate anion. While many amino acids can be potentially converted to PFB derivatives, we have not observed negative ions from amino acids other than 3-nitrotyrosine following purification of the hydrolysate from platelets by HPLC. D e t e r m i n a t i o n of 3 - N i t r o t y r o s i n e in H u m a n Platelets Platelets lose their function when exposed to peroxynitrite,24,25 thus we compared the 3-nitrotyrosine content following exposure of platelet suspension to various nitrating compounds and hormones. Platelets are prepared by centrifugation as described2°'21 and are suspended in I0 ml of HEPES buffer containing glucose (5.56 mM) and sodium bicarbonate (l 1.9 raM). The final concentration of the platelet suspension is 6.8 × 108 ceUs/ml using a Coulter T 540 cell sorter (Coulter Electronics, Hialeah, FL). The protein content of the platelet suspension is determined using the Bradford assay (Bio-Rad). The average protein content is 1.5 mg/ml. One milliliter of the platelet suspension is used for 3-nitrotyrosine analysis following treatment of the platelets with test compounds or controls. The peroxynitrite solution is added to a stirred platelet suspension using a 10-/xl syringe. NO2 gas (l /zl) is taken with a gas-tight syringe and is added immediately to the stirred platelet suspension. NO2CI is generated from nitrite and sodium hypochlorite.21 Platelet samples are stirred for an additional 3-5 rain and are then mixed with 5 ml of ice-cold acetone, vortexed, and kept for l hr at - 2 0 °. I s o l a t i o n of 3 - N i t r o t y r o s i n e The protein precipitate is isolated by low-speed centrifugation and hydrolyzed with 6 M HCI (200/zl) supplemented with I% phenol (v/w) at I l0 ° for 14-18 hr. Prior to hydrolysis, 5 ng of [~3C6]-3-nitrotyrosine is added to the protein precipitate. After cooling, the samples are extracted twice with 200/xl of ethyl acetate to remove lipids. The water phase is dried, dissolved in I0% trichloroacetic acid, and filtered through Cl8 Sep-Pak cartridges (Waters), which are preconditioned with 5 ml methanol and equilibrated with 5 ml water. 3-Nitrotyrosine is eluted quantitatively in 2 ml of 25% methanol/water. The samples are then concentrated and purified by HPLC, derivatized, and analyzed by GCIMS. Control experiments do not show 24 M. A. Moro, V. M. Darley-Usmar, D. A. Goodwin, N. G. Read, R. Zamora-Pino, M. Feelisch, M. W. Radomski, and S. Moncada, Proc. Natl. Acad. Sci. U.S.A. 91, 6702 (1994). 25 C. Boulos, H. Jiang, and M. Balazy, J. Pharmacol. Exp. Ther. 293, 222 (2000).
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3-nitrotyrosine artifacts. A protocol using alkaline protein hydrolysis that avoids nitration artifacts has been reported. 14 D e t e c t i o n of 3 - N i t r o t y r o s i n e in H u m a n Platelets Intact, washed human platelets contain 1.4 -4- 0.6 ng of 3-nitrotyrosine per milligram of platelet proteins (n = 6).21 Exposure of platelets to peroxynitrite (I 0 to 300/zM) produces a dose-dependent increase in platelet 3-nitrotyrosine (4.4- to 535-fold). Decomposed peroxynitrite is without effect (3.4 q- 1.4 vs 1.4 -4- 0.6 ng 3-nitrotyrosine/mg protein). NOz (final concentration, 43 /zM) generates 214 495 ng of 3-nitrotyrosine per milligram of platelet proteins. Treatment of platelets with NaOCI (50/zM) and nitrite (50/zM) generates 4.4 4- 0.3 ng of 3-nitrotyrosine per milligram of protein. Incubations with nitroglycerin (100 #M), nitric oxide (50 IzM), nitrite (50 IzM), and nitrite (50/zM) plus hydrogen peroxide (50/zM) have not increased 3-nitrotyrosine levels. Similarly, platelet agonists (thromhin, ADP, platelet activating factor, arachidonic acid) have no effect on 3-nitrotyrosine levels in platelet proteins. 21 Concluding Remarks Despite complex mechanisms of antioxidant defense, platelets readily undergo nitration by peroxynitrite, which appears to be the most efficient among the compounds tested. In concentrations that are likely to be found in vivo, peroxynitrite caused significant nitration of platelet proteins, which is known to cause loss of platelet function.25 It also appears that protein nitration is unlikely to be induced by hormonal stimulation. Rather, extracellular peroxynitrite or NO2 from other cells or tissues could cause platelet protein nitration. Our methodology for measurements of 3-nitrotyrosine has two useful features. One is the clean method for preparation of the internal standard, 13C-labeled nitrotyrosine. The use of radiolabeled tracer allows preparing its solutions with a precisely determined concentration. The other is a simple one-step preparation of a tri-PFB derivative of 3-nitrotyrosine, which has good chromatographic and mass spectrometric properties. The disadvantage of this method is the rather lengthy purification protocol prior to GC[MS analysis. We are developing this assay further so that the tri-PFB derivative will be analyzed by LC/MS (liquid chromatography/mass spectrometry). More recent methods for 3nitrotyrosine have utilized LC/MS for direct analysis with detection limits of about I n g / m l . 26'27 More advanced techniques of mass spectrometry, such as matrixassisted laser desorpfion ionization time-of-flight mass spectrometry, have been 26 j. S. Althaus, K. R. Schmidt, S. T. Fountain, M. T. Tseng, R. T. Carroll, E Galatsis, and E. D. Hall, Free Radic. Biol. Med. 29, 1085 (2000). 27 D. Yi, B. A. Ingelse, M. W. Duncan, and G. A. Smythe, J. Am. Soc. Mass Spectrom, 11, 578 (2000).
[36]
QUANTITATIVE ANALYSIS OF PROTEIN TYROSINE NITRATION
399
used for the sequencing of proteins containing nitrotyrosine to identify those tyrosine residues that have greater susceptibility for nitration.28'29 Future developments in this area are anticipated to focus on methodologies for the rapid identification and sequencing of nitrated proteins and their metabolism. In addition, methods for 3-nitrotyrosine metabolites found in urine (3-nitrohydroxyphenyllactic acid and 3-nitrohydroxyphenylacetic acid) need to be developed. Our method has the potential of being adapted for measurement of these metabolites. Acknowledgments This research was supported by grants from the American Heart Association, N.Y. State Affiliate (9850104), and the National Institutes of Health (GM 62453).
28 A. Sarver, N. K. Schemer, M. D. Shetlar, and B. W. Gibson, J. Am. Soc. Mass Spectrom. 12, 439
(2001). 29 A. Gaur, P. Kowalski, and M. Balazy, "Proceedings of the 49th A S M S Conference on Mass Spectrometry and AlliedTopics,"Chicago, IL, May 27-31, 2001.
[36] Quantitation and Localization of Tyrosine Nitration in Proteins B y PATRICK S.-Y. W O N G a n d A L B E R T VAN DER VLIET
The formation of reactive oxidized metabolites of nitric oxide (NO') such as peroxynitrite (ONOO-) 1'2 and nitrogen dioxide (NO2") 1'2 is generally believed to play a contributing role in the pathology of inflammatory diseases. In contrast to their precursor nitric oxide (NO'), these reactive nitrogen species (RNS) can covalently modify many classes of biomolecules, thereby potentially inducing functional changes. Important modifications in proteins include the oxidation of amino acid residues such as cysteine, methionine, tryptohan, and tyrosine,3-6 the formation of protein carbonyls,4 and protein fragmentation or cross-linking. More 1 H. Ischiropoulos, Arch. Biochem. Biophys. 356, 1 (1998). 2 j. S. Beckman, Chem. Res. Toxicol. 9, 836 (1996). 3 B. Alvarez, G. Ferrer-Sueta, B. A. Freeman, and R. Radi, J. Biol. Chem. 274, 842 (1999). 4 M. Tien, B. S. Berlett, R. L. Levine, P. B. Chock, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 96, 7809 (1999). 5 W. Wu, Y. Chen, and S. L. Hazen, J. Biol. Chem. 274, 25933 (1999). 6 j. p. Eiserich, M. I-Iristova, C. E. Cross, A. D. Jones, B. A. Freeman, B. Halliwell, and A. van der Vliet, Nature 391, 393 (1998).
METHODSIN ENZYMOLOGY,VOL.359
Copyright2002,ElsevierScience(USA). All rightsreserved. 0076-6879/02$35.00
PEROXYNITRITE
[351
[351 Gas Chromatography/Mass Spectrometry Assay for 3-Nitrotyrosine By MICHAELBALAZY
Introduction Measurements of 3-nitrotyrosine levels in biological samples are being used to evaluate exposure to reactive nitrogen species in vivo. Because protein tyrosine nitration has been detected in many pathophysiological conditions, 1-3 it is thought that potent nitrating compounds are generated during disease processes. A product of nitric oxide (NO) and superoxide, the peroxynitrite (ONOO-) has been shown originally to cause tyrosine nitration.4 Recent work also suggests that other nitrating molecules, such as nitryl chloride and nitrogen dioxide, may be generated in vivo from the oxidation of nitrite by peroxidases. 5'6 Measurement of 3-nitrotyrosine in animal studies, as well as studies involving human subjects, can lead to a better understanding of the basic biochemical principles involved in free radical damage mediated by reactive nitrogen species. Furthermore, pharmacological control of the synthesis of 3-nitrotyrosine-labeled proteins might lead to alleviation of many of the symptoms associated with such damage. One of the factors that inhibit progress in this area is the difficulty of quantifying 3-nitrotyrosine in proteins, as sensitive methods are required to detect small quantities of 3-nitrotyrosine. Methods currently available for the measurement of 3-nitrotyrosine based on competitive binding with immunoglobulins raised against the 3-nitrotyrosine- or 3-nitrotyrosine-containing peptides and on HPLC suffer from a lack of sensitivity and/or specificity. Various immunoassay techniques, including enzyme-linked immunoassay, have been reported to measure 3-nitrotyrosine-containing proteins. 7,8 These assays represent very sensitive means by which one can detect these particular products. Although the antibodies are useful to localize 3-nitrotyrosine in tissues, it is difficult to quantify 3-nitrotyrosine using these techniques. 9'1° Several 1 K. A. Hanafy, J. S. Krumenacker, and E Murad, Med. Sci. Monit. 7, 801 (2001). 2 S. A. Greenacre and H. Ischiropoulos, Free Radic. Res. 3d, 541 (2001). 3 C. Oldreive and C. Rice-Evans, Free Radic. Res. 35, 215 (2001). 4 H. Ischiropoulos, L. Zhu, J. Chen, M. Tsai, J. C. Martin, C. D. Smith, and J. S. Beckman, Arch. Biochem. Biophys. 298, 431 (1992). 5 j. p. Eiserich, M. I-Iristova, C. E. Cross, and A. D. Jones, et al., Nature 391, 393 (1998). 6 L. Gebicka, Acta Biochim. Pol. 46, 919 (1999). 7 j. Khan, D. M. Brennan, N. Bradley, and B. Gao, et al., Biochem. J. 330, 795 (1998). s y. Z. Ye, M. Strong, Z. Q. Huang, and J. S. Beckman, Methods Enzymol. 269, 201 (1996). 9 A. van der Vliet, J. P. Eiserich, H. Kaur, and C. E, Cross, et al., Methods EnzymoL 269, 175 (1996). l0 j. p. Crow and H. Ischiropoulos, Methods Enzymol. 269, 185.
METHODSIN ENZYMOLOGY,VOL.359
Copyright2002,ElsevierScience(USA). All fightsreserved. 0076-6879/02$35.00
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GC/MS OF 3-NITROTYROSINE
391
HPLC assays have been reported that can separate and detect 3-nitrotyrosine with ultraviolet or electrochemical detectors, 1°A1 and detection limits in the range of several micrograms per milligram of protein have been reported. 1° Reduction of 3-nitrotyrosine with dithionite generates 3-aminotyrosine, which can be detected using fluorescence or electrochemical detectors, and enhances the sensitivity 10- to 50-fold relative to the ultraviolet detection. 1°'12 A major disadvantage of the assays based on HPLC is the lack of specificity because artifacts frequently interfere with 3-nitrotyrosine peaks.13 Mass spectrometry is, in principle, an ideal method for detection and quantification of 3-nitrotyrosine because of its sensitivity, specificity, and selectivity. 14 Methods that have been developed mostly rely on converting 3-nitrotyrosine into a more volatile molecule by a series of reactions called derivatization. In this process the carboxyl, hydroxy, and amino groups are converted into an ester, ether, and amide. The final derivative should produce a sharp chromatographic peak and a useful ion for quantification. In an ideal case, a 3-nitrotyrosine derivative that generates a single ion without other fragments will provide the highest sensitivity because it will be converted quantitatively into a detectable ion. This task is difficult to achieve because known 3-nitrotyrosine derivatives produce significant fragmentation largely because they employ several reactions with different chemicals. 14-17 While heptafluorobutyryl and pentafluoropropionyl derivatives of amino acids have been synthesized TM and used for 3-nitrotyrosine quantification, 17,19 it appears that noncharacteristic ions such as C2F5COO-, [C2F5COO2H - F]-, and C2FaCOOare more abundant than ions specific for the amino acid structure.18 In addition, the electron capture mass spectra of these derivatives depend strongly on the amount of sample entering the ion source and, therefore, show considerable variation across the width of the gas chromatographic peak. 18 This could lead to problems in quantitative analysis when working with pentafluoropropionate derivatives. 18 Use of these reagents poses an additional complication because the 3-nitrotyrosine hydroxyl group needs to be further protected with another reagent. 11K. Hensley,M. L. Maidt, Q. N. Pye, C. A. Stewart, M. Wack,T. Tabatabaie, and R. A. Floyd,Anal. Biochem. 251, 187 (1997). 12M. K. Shigenaga,H. H. Lee, B. C. Blount, S. Christen, E. T. Shigeno,H. Yip, and B. N. Ames,Proc. Natl. Acad. Sci. U.S.A. 94, 3211 (1997). t3 H. Kaur, L. Lyras, P. Jenner, and B. Halliwell, J. Neurochem. 70, 2220 (1998). 14M. T. Frost, B. Halliwell, and K. P. Moore, Biochem. J. 345(Pt 3), 453 (2000). t5 E. Schwedhelm,D. Tsikas, E M. Gutzld, and J. C. Frolich,Anal. Biochem. 276, 195 (1999). 16S. Pennathur, V. Jackson-Lewis, S. Przedborski, and J. W. Heinecke, J. Biol. Chem. 274, 34621 (1999). 17j. R. Crowley,K. Yarasheski, C. Leeuwenburgh,J. Turk, and J. W. Heinecker,Anal. Biochem. 259, 127 (1998). 18G. K. C. Low and A. M. Duftield, Biomed. Mass Spectrom. 11, 223 (1984). 19C. Leeuwenburgh,M. M. Hardy, S. L. Hazen, P. Wagner, S. Oh-ishi, U. P. Steinbrecher, and J. W. Heinecke, Z Biol. Chem. 272, 1433 (1997).
392
PEROXYNITRITE
[351
Pentafluorobenzyl (PFB) esters are unique in that they can generate abundant carboxylate anions in negative ion chemical ionization mass spectrometry for compounds such as acidic lipids. 2° We found that 3-nitrotyrosine can be convened into a novel PFB derivative in a single step, and this derivative produces a single ion suitable for a sensitive and quantitative GC/MS assay.21 Chemicals Pentafluorobenzyl bromide (a-bromo-2,3,4,5,6-pentafluorotoluene), diisopropylethylamine, nitronium borofluorate (NO2BF4), and 3-nitro-Ltyrosine are purchased from Aldrich Chemical Co, (Milwaukee, WI) L-4-Hydroxyphenyl-[13C6 ]alanine ([13C6]tyrOsine,isOtOpic abundance >99%; [12C]tyrosine <0.01%) is from Isotec Inc. (Miamisburg, OH) [3,5-3H2]tyrosine (specific activity 59.7 Ci/mmol) is from New England Nuclear (Cambridge, MA) Peroxynitrite has been synthesized in a quenched-flow reactor as described previously22 All solvents are of chromatographic purity from Fisher Scientific. P r e p a r a t i o n of 13C-Labeled 3 - N i t r o t y r o s i n e a s a n I n t e r n a l S t a n d a r d for M a s s S p e c t r o m e t r y One hundred micrograms of [13C6]tyrosine and l0/zCi of 3H-labeled tyrosine (as separate solutions in water/ethanol, 98 : 2) are placed in a dry glass tube, and the solvent is evaporated under vacuum to dryness. The residue is dissolved in 100/zl of a l M solution of NO2BF4 in 0.I M HCI with vigorous vortexing. The NO2BF4 solution should be prepared shortly before use. The tube is then capped with a Teflon cap and placed in a Pierce laboratory heater, with temperature adjusted to 70 °, for 60 min with occasional shaking. After the reaction mixture is cooled, aliquots (20 #l) are injected into a C18 column (150 x 4.6 mm, Beckman, Fullerton, CA) installed in a HPI050 HPLC system (Hewlett-Packard, Palo-Alto, CA). Samples are eluted with a gradient of acetonitrile in water (0 to 100% in 20 rain) at a flow rate of 1 ml/min. The reaction products can be detected by a radioactivity monitor (Packard, Meriden, CT) with an Ecolite (ICN, Costa Mesa, CA) scintillation cocktail and/or a UV detector (205 nm). The effluent from the column is collected into glass tubes as 1-ml fractions using a Gilson FC203B fraction collector. The radiolabeld products in these fractions are identified by scintillation counting of 50-/zl aliquots. [13C6]-3-Nitrotyrosine is collected in a single fraction elufing at 5.5-6.5 rain. The final stock solution is obtained in 5 ml 20 M. Balazy, J. Biol. Chem. 266, 23561 (1991). 21 H. Jiang and M. Balazy, Nitric Oxide 2, 350 (1998). 22 M. Balazy, Pol. J. Pharmacol. 46, 593 (1994).
[35]
GC/MS oF 3-NITROTYROSINE
393
of 0.1 M HCI. The concentration of this stock solution is finally determined via scintillation counting of radioactivity in a 100-/xl aliquot. Finally, a solution having a concentration of 0.1 ng [13Cr]-3-nitrotyrosine/ml is prepared. Our attempts to synthesize [13C6]-3-nitrotyrosine using tetranitromethane23 have not been successful. We experienced low yields and difficulty removing a major closely eluting contaminant by HPLC chromatography. Treatment of tyrosine with NO2BF4 gives typical yields of 3-nitrotyrosine in the range of 80-90%. A minor product, 3-nitrohydroxyphenyllactic acid (10-20%), is well separated from 3-nitrotyrosine (Fig. 1). Thus the reaction of NO2BF4 with tyrosine occurs via electrophilic aromatic nitration, whereas substitution of the amino group by a hydroxyl, possibly via an unstable diazonium salt intermediate, is a minor process. Because nitration occurs at carbons labeled with tritium, this reaction also generates a tritium-labeled hydroborotetrafiuoric acid by-product, which elutes as an early peak of radioactivity (Fig. 1). In our experience, use of the radioactive tracer, [3,5-3H2]tyrosine, is essential to monitor the progress of the reaction, as well as in establishing the final concentrations of [13C6]-3-nitrotyrosine solutions. It is also important to note that because one tritium was removed and replaced by an NO2 group, the specific activity of the final product decreases by 50% relative to the specific activity of the original radiolabeled tyrosine. This observation should be taken into account when calculating the final concentration of the [13C6]-3nitrotyrosine stock solution. Analysis by GC/MS of the product eluting at 5.56.5 min confirmed the structure of pure 13C-labeled 3-nitrotyrosine (Fig. 1). P r e p a r a t i o n of P e n t a f l u o r o b e n z y l Derivative of 3 - N i t r o t y r o s i n e The PFB derivative of 3-nitrotyrosine is prepared by the addition of PFB bromide (40/zl) and diisopropylethylamine (40/xl) followed by vigorous mixing and heating at 70 ° for 40 rain. Both reagents for derivatization are prepared as I0% solutions in acetonitrile. Derivatization is terminated by the evaporation of reagents under a genre stream of nitrogen. The residue is then dissolved in 50/zl of methanol and is subjected to purification by HPLC using an acetonitrile gradient in water (50 to I00% in 20 rain; Cl8 column, 150 × 4.6 nun, Beckman). The PFB derivative of 3-nitrotyrosine is typically collected at 13-14 rain. After evaporation of the solvent under vacuum, samples are dissolved in 50/zl of n-decane and analyzed by GC/MS (Fig. 2). The reaction of PFB bromide with 3-nitrotyrosine in the presence of catalytic amounts of diisopropylethylamine quantitatively generates a new derivative (Fig. 3). Treatment of this derivative with a siliconizing compound, BSTFA [N,O-bis(trimethylsilyl)trifluoroacetan~de], does not alter its mass spectrum, indicating that, in addition to the carboxyl, both the hydroxyl and the amino group also reacted with PFB bromide and, therefore, could not be converted into a TMS 23M. Sokolovsky,J. F. Riordan,and B. L. Vallee,Biochemistry5, 3582 (1966).
394
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GC/MS OF 3-NITROTYROSINE
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(trimethylsilyl) derivative. A hydroxyl vicinal to a nitro group in 3-nitrotyrosine has a pKa of approximately 7.5, which makes it sufficiently acidic to react quantitatively with PFB bromide yielding a PFB ether. This is an uncommon reaction, as primary and secondary alcohols usually do not react with PFB bromide. The heptafluoro derivative of 3-nitrotyrosine requires additional derivatization of the hydroxyl with TBDMS prior to GC/MS analysis. 14 GC/MS Analysis of 3-Nitrotyrosine PFB Ester Analysis can be performed on a suitable GC/MS system equipped with a chemical ionization source and negative ion optics. We have used a HP5989A mass spectrometer interfaced to a HP 5890 gas chromatograph (Hewlett-Packard). Samples (I/zl) are injected with an autoinjector into a capillary chromatographic column (DB-I fused silica, 10 m, 0.25 mm i.d., 0.25 ]zm film thickness, J&W Scientific, Rancho Cordova, CA) via a splitless injector having a temperature of 250 °. The oven temperature is programmed from 150 to 300 ° at 30 °/min during the analysis. Helium is used as the carder gas with a linear velocity of 4 m/sec. Electron-capture ionization is carded out with methane as a moderating gas delivered to produce a pressure in the ion source of 2.8 Torr. The other mass spectrometer parameters are as follows: ion source temperature, 200°; electron energy, 220 eV; and transfer line temperature, 250 ° . Selected ion monitoring is used to record ion abundances at m/z 585 and 591, which correspond to the PFB derivatives of 3-nitrotyrosine and [13C6]-3-nitrotyrosine (internal standard), respectively. Areas under chromatographic peaks defined for ions at m/z 585 and 591 are obtained from GC/MS integration data (Chemstation software). A standard curve is prepared by mixing 3-nitrotyrosine (0 to 40 ng) and [13C6]-3-nitrotyrosine (5 ng) followed by derivatization, purification, and GC/MS analyses as described earlier. The amount of 3-nitrotyrosine in the sample is calculated from the regression line that shows good linearity ( r 2 = 0.999) when plotted as a function of 3-nitrotyrosine concentration, and the ratio of the peak areas is generated by ions m/z 585 and 591 (Fig. 2). A detection limit of 10 pg for the 3-nitrotyrosine PFB derivative has been achieved with a signal-to-noise ratio greater than 3.
[35]
GC/MS OF 3-NITROTYROSINE
397
The tri-PFB derivative of 3-nitrotyrosine shows a sharp and symmetrical chromatographic peak and a simple mass spectrum (Figs. 1 and 2). A prominent ion at m/z 591 corresponds to a carboxylate anion of [13C6]-3-nitrotyrosine and originates from the electron-capture-mediated loss of the PFB radical typically observed in these esters. Formation of such an anion is a favorable process because of its high efficiency and intrinsic stability of the carboxylate anion. While many amino acids can be potentially converted to PFB derivatives, we have not observed negative ions from amino acids other than 3-nitrotyrosine following purification of the hydrolysate from platelets by HPLC. D e t e r m i n a t i o n of 3 - N i t r o t y r o s i n e in H u m a n Platelets Platelets lose their function when exposed to peroxynitrite,24,25 thus we compared the 3-nitrotyrosine content following exposure of platelet suspension to various nitrating compounds and hormones. Platelets are prepared by centrifugation as described2°'21 and are suspended in I0 ml of HEPES buffer containing glucose (5.56 mM) and sodium bicarbonate (l 1.9 raM). The final concentration of the platelet suspension is 6.8 × 108 ceUs/ml using a Coulter T 540 cell sorter (Coulter Electronics, Hialeah, FL). The protein content of the platelet suspension is determined using the Bradford assay (Bio-Rad). The average protein content is 1.5 mg/ml. One milliliter of the platelet suspension is used for 3-nitrotyrosine analysis following treatment of the platelets with test compounds or controls. The peroxynitrite solution is added to a stirred platelet suspension using a 10-/xl syringe. NO2 gas (l /zl) is taken with a gas-tight syringe and is added immediately to the stirred platelet suspension. NO2CI is generated from nitrite and sodium hypochlorite.21 Platelet samples are stirred for an additional 3-5 rain and are then mixed with 5 ml of ice-cold acetone, vortexed, and kept for l hr at - 2 0 °. I s o l a t i o n of 3 - N i t r o t y r o s i n e The protein precipitate is isolated by low-speed centrifugation and hydrolyzed with 6 M HCI (200/zl) supplemented with I% phenol (v/w) at I l0 ° for 14-18 hr. Prior to hydrolysis, 5 ng of [~3C6]-3-nitrotyrosine is added to the protein precipitate. After cooling, the samples are extracted twice with 200/xl of ethyl acetate to remove lipids. The water phase is dried, dissolved in I0% trichloroacetic acid, and filtered through Cl8 Sep-Pak cartridges (Waters), which are preconditioned with 5 ml methanol and equilibrated with 5 ml water. 3-Nitrotyrosine is eluted quantitatively in 2 ml of 25% methanol/water. The samples are then concentrated and purified by HPLC, derivatized, and analyzed by GCIMS. Control experiments do not show 24 M. A. Moro, V. M. Darley-Usmar, D. A. Goodwin, N. G. Read, R. Zamora-Pino, M. Feelisch, M. W. Radomski, and S. Moncada, Proc. Natl. Acad. Sci. U.S.A. 91, 6702 (1994). 25 C. Boulos, H. Jiang, and M. Balazy, J. Pharmacol. Exp. Ther. 293, 222 (2000).
398
PEROXYNITRITE
[35]
3-nitrotyrosine artifacts. A protocol using alkaline protein hydrolysis that avoids nitration artifacts has been reported. 14 D e t e c t i o n of 3 - N i t r o t y r o s i n e in H u m a n Platelets Intact, washed human platelets contain 1.4 -4- 0.6 ng of 3-nitrotyrosine per milligram of platelet proteins (n = 6).21 Exposure of platelets to peroxynitrite (I 0 to 300/zM) produces a dose-dependent increase in platelet 3-nitrotyrosine (4.4- to 535-fold). Decomposed peroxynitrite is without effect (3.4 q- 1.4 vs 1.4 -4- 0.6 ng 3-nitrotyrosine/mg protein). NOz (final concentration, 43 /zM) generates 214 495 ng of 3-nitrotyrosine per milligram of platelet proteins. Treatment of platelets with NaOCI (50/zM) and nitrite (50/zM) generates 4.4 4- 0.3 ng of 3-nitrotyrosine per milligram of protein. Incubations with nitroglycerin (100 #M), nitric oxide (50 IzM), nitrite (50 IzM), and nitrite (50/zM) plus hydrogen peroxide (50/zM) have not increased 3-nitrotyrosine levels. Similarly, platelet agonists (thromhin, ADP, platelet activating factor, arachidonic acid) have no effect on 3-nitrotyrosine levels in platelet proteins. 21 Concluding Remarks Despite complex mechanisms of antioxidant defense, platelets readily undergo nitration by peroxynitrite, which appears to be the most efficient among the compounds tested. In concentrations that are likely to be found in vivo, peroxynitrite caused significant nitration of platelet proteins, which is known to cause loss of platelet function.25 It also appears that protein nitration is unlikely to be induced by hormonal stimulation. Rather, extracellular peroxynitrite or NO2 from other cells or tissues could cause platelet protein nitration. Our methodology for measurements of 3-nitrotyrosine has two useful features. One is the clean method for preparation of the internal standard, 13C-labeled nitrotyrosine. The use of radiolabeled tracer allows preparing its solutions with a precisely determined concentration. The other is a simple one-step preparation of a tri-PFB derivative of 3-nitrotyrosine, which has good chromatographic and mass spectrometric properties. The disadvantage of this method is the rather lengthy purification protocol prior to GC[MS analysis. We are developing this assay further so that the tri-PFB derivative will be analyzed by LC/MS (liquid chromatography/mass spectrometry). More recent methods for 3nitrotyrosine have utilized LC/MS for direct analysis with detection limits of about I n g / m l . 26'27 More advanced techniques of mass spectrometry, such as matrixassisted laser desorpfion ionization time-of-flight mass spectrometry, have been 26 j. S. Althaus, K. R. Schmidt, S. T. Fountain, M. T. Tseng, R. T. Carroll, E Galatsis, and E. D. Hall, Free Radic. Biol. Med. 29, 1085 (2000). 27 D. Yi, B. A. Ingelse, M. W. Duncan, and G. A. Smythe, J. Am. Soc. Mass Spectrom, 11, 578 (2000).
[36]
QUANTITATIVE ANALYSIS OF PROTEIN TYROSINE NITRATION
399
used for the sequencing of proteins containing nitrotyrosine to identify those tyrosine residues that have greater susceptibility for nitration.28'29 Future developments in this area are anticipated to focus on methodologies for the rapid identification and sequencing of nitrated proteins and their metabolism. In addition, methods for 3-nitrotyrosine metabolites found in urine (3-nitrohydroxyphenyllactic acid and 3-nitrohydroxyphenylacetic acid) need to be developed. Our method has the potential of being adapted for measurement of these metabolites. Acknowledgments This research was supported by grants from the American Heart Association, N.Y. State Affiliate (9850104), and the National Institutes of Health (GM 62453).
28 A. Sarver, N. K. Schemer, M. D. Shetlar, and B. W. Gibson, J. Am. Soc. Mass Spectrom. 12, 439
(2001). 29 A. Gaur, P. Kowalski, and M. Balazy, "Proceedings of the 49th A S M S Conference on Mass Spectrometry and AlliedTopics,"Chicago, IL, May 27-31, 2001.
[36] Quantitation and Localization of Tyrosine Nitration in Proteins B y PATRICK S.-Y. W O N G a n d A L B E R T VAN DER VLIET
The formation of reactive oxidized metabolites of nitric oxide (NO') such as peroxynitrite (ONOO-) 1'2 and nitrogen dioxide (NO2") 1'2 is generally believed to play a contributing role in the pathology of inflammatory diseases. In contrast to their precursor nitric oxide (NO'), these reactive nitrogen species (RNS) can covalently modify many classes of biomolecules, thereby potentially inducing functional changes. Important modifications in proteins include the oxidation of amino acid residues such as cysteine, methionine, tryptohan, and tyrosine,3-6 the formation of protein carbonyls,4 and protein fragmentation or cross-linking. More 1 H. Ischiropoulos, Arch. Biochem. Biophys. 356, 1 (1998). 2 j. S. Beckman, Chem. Res. Toxicol. 9, 836 (1996). 3 B. Alvarez, G. Ferrer-Sueta, B. A. Freeman, and R. Radi, J. Biol. Chem. 274, 842 (1999). 4 M. Tien, B. S. Berlett, R. L. Levine, P. B. Chock, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 96, 7809 (1999). 5 W. Wu, Y. Chen, and S. L. Hazen, J. Biol. Chem. 274, 25933 (1999). 6 j. p. Eiserich, M. I-Iristova, C. E. Cross, A. D. Jones, B. A. Freeman, B. Halliwell, and A. van der Vliet, Nature 391, 393 (1998).
METHODSIN ENZYMOLOGY,VOL.359
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