Title: Specific Nitration at Tyrosine 430 Revealed by High Resolution Mass Spectrometry as Basis for Redox Regulation of Bovine Prostacyclin Synthase
Abstract: Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI2 synthase) with increasing concentrations of peroxynitrite (PN) up to 250 μm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 μm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 μm PN, PGI2 synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427–430 of PGI2 synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI2 synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide. Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI2 synthase) with increasing concentrations of peroxynitrite (PN) up to 250 μm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 μm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 μm PN, PGI2 synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427–430 of PGI2 synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI2 synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide. peroxynitrite (oxoperoxonitrate (1−)) prostacyclin prostacyclin synthase 3-nitrotyrosine prostaglandin endoperoxide 6-keto-prostaglandin F1α enzyme immunoassay nitric oxide phosphate-buffered saline bovine serum albumin bacterial monooxygenase-3 from Bacillus megaterium (CYP 102) Fourier transform ion cyclotron resonance high performance liquid chromatography phenylthiohydantoin matrix-assisted laser desorption ionization time-of-flight N-(9-fluorenyl)methoxycarbonyl The nitration of tyrosine residues in proteins has become a well recognized reaction, but has been heavily disputed with regard to the mechanisms involved and its physiological and/or pathophysiological significance (1Cohen R.A. Prog. Cardiovasc. Dis. 1995; 38: 105-128Crossref PubMed Scopus (261) Google Scholar, 2Moncada S. Vane J.R. Pharmacol. Rev. 1979; 30: 293-331Google Scholar, 3Moncada S. Vane J.R. N. Engl. J. Med. 1979; 300: 1142-1147Crossref PubMed Scopus (914) Google Scholar, 4Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 5Zou M.H. Jendral M. Ullrich V. Br. J. Pharmacol. 1999; 126: 1283-1292Crossref PubMed Scopus (83) Google Scholar). Peroxynitrite (PN)1 generated from nitric oxide (NO) and superoxide (O2⨪) can react with Tyr or Tyr-containing proteins under formation of 3-nitrotyrosine (3-NT) (6Crow J.P. Beckman J.S. Adv. Pharmacol. 1995; 34: 17-43Crossref PubMed Scopus (289) Google Scholar, 7Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar, 8Viner R.I. Williams T.D. Schöneich C. Biochemistry. 1999; 38: 12408-12415Crossref PubMed Scopus (211) Google Scholar) but in general the required concentrations are higher than expected to occur in vivo. Pfeiffer and Mayer (9Pfeiffer S. Mayer B. J. Biol. Chem. 1998; 273: 27280-27285Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 10Pfeiffer S. Schmidt K. Mayer B. J. Biol. Chem. 2000; 275: 6346-6352Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (100) Google Scholar) have even questioned the significance of PN as a cellular nitrating agent and have proposed nitrite/hydrogen peroxide as an alternative pathway with myeloperoxidase as a catalyst (11Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Pfeiffer S. Lass A. Schmidt K. Mayer B. FASEB J. 2001; 15: 2355-2364Crossref PubMed Scopus (100) Google Scholar), which may indeed apply for certain proteins. In the case of PN it has not been considered that PN can be activated by transition metal ions that may then catalyze the self-nitration of metalloproteins at low PN levels. We have recently provided evidence for this reaction for heme-thiolate (P450) proteins (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google Scholar, 14Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, 15Heinz K. Dünstl G. Bachschmid M. Daiber A. Nüsing R. Ullrich V. Nitric Oxide. 2002; 6: 400Google Scholar) that therefore may serve as a model for the P450 protein PGI2 synthase. PGI2 synthase was inactivated by micromolar PN concentrations (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar, 17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar) but also by a continuous generation of NO and O2⨪ from SIN-1 (18Zou M.H. Klein T. Pasquet J.-P. Ullrich V. J. Biochem. 1998; 336: 507-512Crossref Scopus (64) Google Scholar). In cellular systems the inhibition of nitration by a NO synthase inhibitor and polyethylene-glycolated superoxide dismutase provided evidence for the involvement of PN, whereas nitrite was ineffective (18Zou M.H. Klein T. Pasquet J.-P. Ullrich V. J. Biochem. 1998; 336: 507-512Crossref Scopus (64) Google Scholar). Because NO and PGI2 are important for the endothelial barrier function the formation of PN and the nitration of PGI2 synthase could play a role in the process of endothelial activation for adhesion and emigration of white blood cells into the tissue (19Ullrich V. Zou M.H. Bachschmid M. Biochim. Biophys. Acta. 2001; 1532: 1-14Crossref PubMed Scopus (30) Google Scholar). Interestingly, PGI2 synthase was found localized to the caveolae-like endothelial NO synthase (20Spisni E. Griffoni C. Santi S. Riccio M. Marulli R. Bartolini G. Toni M. Ullrich V. Tomasi V. Exp. Cell Res. 2001; 266: 31-43Crossref PubMed Scopus (72) Google Scholar) and hence PN formation may occur in close vicinity to PGI2 synthase. This localization in a "quasi-extracellular" compartment may be a further important factor for efficient nitration by low concentrations of PN. Beyond this physiological background no proof for the molecular basis of enzyme inhibition has been hitherto obtained by identification of nitrated tyrosine. Substrate analogs of prostaglandin-endoperoxide have been recently shown to inhibit the nitration (17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar), which suggested a proximity to the heme attached to the protein by the Cys-441 residue (21Hatae T. Hara S. Yokoyama C. Yabuki T. Inoue H. Ullrich V. Tanabe T. FEBS Lett. 1996; 389: 268-272Crossref PubMed Scopus (34) Google Scholar, 22Shyue S. Ruan K. Wang L. Wu K. J. Biol. Chem. 1997; 272: 3657-3662Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 23Ullrich V. Brugger R. Angew. Chem. Int. Ed. Engl. 1994; 33: 1911-1919Crossref Scopus (73) Google Scholar); however, previous attempts have been unsuccessful to detect and identify the nitrated tyrosine. In this study we present molecular evidence for the specific nitration of bovine PGI2 synthase at tyrosine 430 by high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry (24Rossier J.S. Youhnovski N. Lion N. Damoc E. Reymond F. Girault H.H. Przybylski M. Angew. Chem. Int. Ed. Engl. 2003; 42: 53-58Crossref Scopus (47) Google Scholar), and the presence of 3-NT upon extended Pronase digestion. We further show an unusually slow digestion by thermolysin, presumably because of a tight fold around the heme, to release a tetrapeptide by an unexpected specific cleavage adjacent to the nitrated tyrosine residue. Pronase from Streptomyces griseus(lyophilized powder) was obtained from Roche Molecular Diagnostics. Thermolysin, type X from Bacillus thermoproteolyticus rokkowas purchased from Sigma. All other chemicals were of analytical grade or highest purity available. PN was a gift from Dr. Koppenol (ETH Zürich, Switzerland) and was synthesized from NO and potassium superoxide according to Kissner et al. (25Kissner R. Nauser T. Bugnon P. Lye P.G. Koppenol W.H. Chem. Res. Toxicol. 1997; 10: 1285-1292Crossref PubMed Scopus (554) Google Scholar). P450BM-3 (CYP 102), a F87Y variant from Bacillus megaterium was a kind gift from J. A. Peterson (Southwestern Medical School, Dallas, TX) and was purified as described (26Graham-Lorence S. Truan G. Peterson J.A. Falck J.R. Wei S. Helvig C. Capdevila J.H. J. Biol. Chem. 1997; 272: 1127-1135Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). A rabbit polyclonal antibody against PGI2 synthase was produced according to Siegle et al. (27Siegle I. Nüsing R. Brugger R. Sprenger R. Zecher R. Ullrich V. FEBS Lett. 1994; 347: 221-225Crossref PubMed Scopus (20) Google Scholar). A mouse monoclonal antibody against 3-NT (anti-NT, clone 1A6) was obtained from Upstate Biotechnology (Hamburg, Germany) as a stock solution of 1 mg/ml. Secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG) were obtained from Pierce (stock solutions 0.8 mg/ml). The enhanced chemiluminescence (ECL) kit and nitrocellulose transfer membranes (Hybond C, pore size 0.5 μm) were purchased from Amersham Biosciences. PGH2 was obtained from Cayman Chemical (Ann Arbor, MI). Endothelial and smooth muscle layers from 8 to 10 freshly received bovine aorta were isolated by dissection at 4 °C, rapidly frozen in liquid nitrogen, and stored at −70 °C. Frozen strips were homogenized at 0–4 °C in a Waring blender in 100 mm K-phosphate buffer, pH 7.5, containing 1 mm EDTA, 0.1 mm dithiothreitol, 0.1 mm butylated hydroxytoluene, and 44 mg/liter phenylmethylsulfonyl fluoride. The microsomal fraction was obtained by centrifugation as described (28Ullrich V. Castle L. Weber P. Biochem. Pharmacol. 1981; 30: 2033-2036Crossref PubMed Scopus (82) Google Scholar) to a final volume of 75–100 ml, with a protein concentration of 10–20 mg/ml. The homogenization buffer contained 50 mm K2HPO4, pH 7.5, without additional protease inhibitors. Reaction with PN was carried out with microsomes because active enzyme is required for nitration and further purification steps involving denaturating detergents partially inactivate the protein. PN (10 μl) at a defined concentration was quickly added by thorough Vortex mixing to an ice-cold microsomal suspension (990 μl, total protein concentration 1 mg/ml in 50 mm K-phosphate buffer, pH 7.5). Controls were treated with decomposed PN (24 h at room temperature). The activity for PGI2 formation of PN-treated microsomes was tested by incubation of 100 μl of microsomal suspension (1 mg of protein/ml) in 100 mm potassium phosphate buffer, pH 7.4, with PGH2 (1 μg) for 1 min at 20 °C. To avoid cross-reactivities with PGH2 degradation products the incubation mixture was stopped with 20 μl of 1 m citric acid and extracted two times with 300 μl of ethyl acetate and separated by TLC (Silica Gel 60, Merck, Darmstadt, Germany); solvent: ethyl acetate:2,2,4-trimethylpentane:acetic acid:water, 10:50:20:100). 6-Keto-PGF1α was identified by an iodine-stained reference (RF value about 0.18). The area of 6-keto-PGF1α was excised, extracted with ethyl acetate, and evaporated to complete dryness. After addition of 100 μl of PBS three dilutions of 1:100, 1:1000, and 1:10000 were prepared and tested by EIA (Assay Designs Inc., BioTrend, Köln, Germany) according to the manufacturer's protocol. The microsomal samples were treated for 5 min at 95 °C with Laemmli buffer and separated by 8% (v/v) SDS-PAGE (30 mA, 1 h). The proteins were then transferred onto a nitrocellulose membrane by a semidry blot procedure using a constant current of 50 mA for 90 min. The blotting buffer contained 48 mm Tris, 39 mm glycine, 20% (v/v) methanol, and 0.037% (w/v) SDS. Transfer efficiency of proteins was examined by staining with 0.1% Ponceau S in 5% (v/v) acetic acid. After destaining in PBS, the membrane was blocked with 5% (w/v) milk powder in PBS, pH 7.4, for 2 h at room temperature or at 4 °C overnight. The membrane was then incubated for 2 h with a polyclonal antibody against PGI2 synthase (1 μg/ml PBS). After repeated washing with PBS, 0.1% Tween 20 the membrane was incubated for 45 min with a horseradish peroxidase-conjugated goat anti-rabbit antibody at a dilution of 1:7500 for 45 min, and ECL was used for detection of antibody binding according to the manufacturer's instructions. Prior to staining with a second antibody the membrane was stripped by incubation in stripping buffer (62.5 mm Tris-HCl, pH 6.7, 2% (w/v) SDS, 100 mm 2-mercaptoethanol) under gentle shaking for 60 min at 70 °C. After washing and blocking, the membrane was incubated with a mouse monoclonal antibody against 3-NT at a dilution of 1 μg/ml, followed by a horseradish peroxidase-conjugated goat anti-mouse antibody at a dilution of 1:7500. PGI2 synthase samples were always stained first with the PGI2 synthase antibody, then stripped one or two times before staining with the NT antibody, to ensure complete denaturation for recognition of nitrated protein. Separation of PGI2 synthase from microsomal membranes and solubilization was performed by adding 1% (v/v) Triton X-100 to aortic microsomes. The suspension was stirred for 2 h at 4 °C, then centrifuged for 1 h at 100,000 × g to yield a clear yellow supernatant. Because SDS-PAGE is hampered by the high actin concentration (about 80–90% of total protein) in microsomes, actin was partially removed by precipitation with 15 mmCaCl2 for 1 h at 4 °C and centrifugation of the precipitate for 5 min at 10,000 × g. Because Triton X-100 interfered with SDS-PAGE in subsequent purification steps, detergent was removed by extracting with chloroform and Vortex mixing for a few seconds (29Horikawa S. Ogawara H. Anal. Biochem. 1979; 97: 116-119Crossref PubMed Scopus (25) Google Scholar). After centrifugation for 30 min at 3000 rpm, the organic and aqueous phases were recovered. Proteins precipitated at the interphase as a solid white layer, and remaining chloroform was removed by evaporation. Proteins were then solubilized in SDS-containing electrophoresis buffer (25 mm Tris, 192 mm glycine, 0.1% (w/v) SDS). Proteins were treated with Laemmli sample buffer for 5 min at 95 °C and then separated by 10% (v/v) SDS-PAGE (100 mA, 5 h) on preparative gels (160 × 165 mm, thickness 1.5 mm). Reverse staining by imidazole:zinc was applied for visualization of bands (30Fernandez-Patron C. Castellanos-Serra L. Rodriguez P. BioTechniques. 1992; 12: 564-573PubMed Google Scholar). Gels were equilibrated for 15 min in 100 ml of 0.2 m imidazole in water with gentle shaking, and then placed for 1 min in 100 ml of 0.3 m ZnCl2. The staining solution was removed when the background became deep white showing the transparent protein bands. The band containing PGI2 synthase was excised with a razor blade, immersed in 2% citric acid solution 2–3 times (10 min) to remove zinc ions from the gel matrix, and washed several times with SDS-PAGE buffer. Recovery of PGI2 synthase was performed by electroelution (ELUTRAP, Schleicher & Schuell, Dassel, Germany) (31Jacobs E. Clad A. Anal. Biochem. 1986; 154: 583-589Crossref PubMed Scopus (87) Google Scholar). Protein bands were cut to slices, placed in the elution chamber and covered with SDS-PAGE buffer (25 mm Tris, 192 mm glycine, 0.1% (w/v) SDS). A voltage of 200 V corresponding to the ∼50 mA current was then applied. After an 8-h elution period proteins accumulated inside a trap (volume 800 μl) between two membranes were collected with a pipette. Protein solutions were finally concentrated with an Ultrafree-4 Centrifugal filter unit (Millipore Corp.) to a 5 μm solution (0.28 mg/ml). Protein concentrations were determined with the Bio-Rad DC assay (Bio-Rad). Samples of 100 μl of electroeluted PGI2 synthase were treated with different concentrations of PN (0, 10, 50, 100, and 250 μm), heated for 10 min to 95 °C, and then mixed with 10 mm CaCl2 to stabilize proteases. After addition of Pronase (1 mg/ml) the samples were incubated for 24 h at 40 °C, then another 1 mg/ml Pronase was added and incubated again for 24 h at 50 °C. Digestion was repeated with a third and fourth portion (0.5 mg/ml) for 12 h at 50 °C. Prior to HPLC separation the samples were filtered with the 10-kDa MICROCON centrifugal filter device (Millipore Corp.) by centrifuging for 30 min at 10,000 × g. Products were analyzed on a Jasco HPLC system consisting of a PU-980 pump, a Jasco UV-1575 and Spectra Physics spectra focus UV-visible detector, and a LG-980-02 low pressure mixing unit. A C18 Nucleosil 100-5 250 × 4.6 column from Macherey & Nagel (Düren, Germany) was used with a mobile phase gradient (0–15 min, 0% (v/v) B; 15–30 min, 0–90% (v/v) B; 30–40 min, 90% (v/v) B (A: 0.1% (v/v) trifluoroacetic acid, pH 2, B: 80% (v/v) acetonitrile with 0.08% (v/v) trifluoroacetic acid)). The flow rate was 1 ml/min and sample aliquots of 100 μl were injected. Tyrosine, phenylalanine, tryptophan, and 3-NT were identified and quantified at 270 and 360 nm by internal and external standards. The retention time of 3-NT was 12 min. As a control 3-NT was reduced with sodium dithionite to 3-aminotyrosine. Because of the large amount of SDS in the electroeluted protein solution (>1% SDS) in-gel digestion was more suitable than digestion in solution. Protein solutions (about 0.5 nmol of PGI2 synthase isolated from treated (25 μm PN) or untreated microsomes) were incubated in Laemmli buffer for 5 min at 95 °C and separated by SDS-PAGE on a "Novex" 8% Tris glycine gel (10 wells, Invitrogen; 30 mA, 1 h). Protein bands were visualized by reverse staining with imidazole:zinc as described above. Proteolytic digestion in the gel matrix was carried out according to the procedure of Shevchenko et al. (32Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1283) Google Scholar). The protein bands were excised from the gel, cut to pieces and washed with 2% citric acid, then with water to remove staining dye, gel buffers, and SDS, and dried at room temperature in a vacuum centrifuge. The washing step was repeated by dehydration of the gel pieces and discarding the solution. After shrinking by vacuum centrifugation the gel pieces were reswollen in 200 μl of digest solution containing 50 mm Tris, pH 8.0, 5 mm CaCl2, 10% (v/v) acetonitrile, and 25 ng/μl thermolysin, and the supernatant was removed. Proteolytic digestion was carried out for 24 h at 50 °C under gentle shaking. Peptides were extracted several times with 0.1% trifluoroacetic acid:acetonitrile for 24 h, lyophilized to dryness, and analyzed on the above described HPLC system. A C18 Nucleosil 100-3 125 × 4.6 column from Macherey & Nagel was used with a mobile phase gradient (0–5 min, 0% (v/v) B; 5–50 min, 0–60% (v/v) B (A: 0.1% (v/v) trifluoroacetic acid in water, pH 2, B: 80% (v/v) acetonitrile with 0.08% (v/v) trifluoroacetic acid)), at a flow rate of 0.8 ml/min. Peptide fragments were detected at 220 and 365 nm; peaks showing a strong absorption at 365 nm were collected and lyophilized for further analysis. Sequence determination of the isolated peptide was achieved by Edman amino acid sequencing. NH2-terminal Edman degradation was performed on an Procise HT sequencing system, model 494 (PerkinElmer Life Sciences, Weiterstadt, Germany), fitted with an online, narrow-bore HPLC-based amino acid analyzer that utilized a 220 × 2.1-mm C18reversed-phase column held at 55 °C in a column heater oven. Released phenylthiohydantoin (PTH)-derivatives from each cycle were separated under the recommended binary gradient conditions using 3.5% tetrahydrofuran in water (buffered with sodium phosphate, pH 4.5; solvent A) and 10% 2-propanol in acetonitrile (unbuffered; solvent B). Prior to sequence determination, samples of peptides were applied to a biobrene-treated glass fiber disk and allowed to dry in a stream of argon. Reagents, operating software, and protocols were used as described from the instrument manufacturer. Chromatographic identification of the UV signals was done by reference to the retention times and the absorbance of a PTH standard run. PTH-derivatives display characteristic UV spectra with an absorbance maximum at 269 nm. The nitrated tetrapeptide LKNY(nitro) was synthesized on a semiautomated peptide synthesizer (EPS-221, Abimed) using solid-phase peptide synthesis Fmoc chemistry methods (33Michels J. Geyer A. Mocanu V. Welte W. Burlingame A.L. Przybylski M. Protein Sci. 2002; 11: 1565-1574Crossref PubMed Scopus (10) Google Scholar) with all chemicals of analytical grade or highest available purity. Fmoc amino acids, NovaSyn TGR resin, PyBop, and other reagents were obtained from Novabiochem (Laufelfingen, Switzerland). To synthesize the peptide with COOH-terminal 3-NT carboxamide the TGR resin was employed with 40 min coupling time and 5 min deprotection in 20% piperidine inN,N-dimethylformamide. Purification of the peptide was performed with preparative HPLC on a Grom-Sil ODS-4Me column. High resolution mass spectrometry was performed with a 7T Bruker Daltonik (Bremen, Germany) Apex II FT-ICR mass spectrometer equipped with an actively shielded 7.0 tesla superconducting magnet (Magnex, Oxford, UK), an APOLLO (Bruker Daltonik) electrospray ionization source and nano-electrospray system, an API1600 ESI control unit, and a UNIX based Silicon Graphics O2 work station. Details of the instrumental conditions of ESI-FT-ICR-MS were as previously reported (34Bauer S.H. Wiechers M.F. Bruns K. Przybylski M. Stuermer C.A. Anal. Biochem. 2001; 298: 25-31Crossref PubMed Scopus (19) Google Scholar). The mass spectra were obtained by collecting 32–124 single scans. Experimental conditions were: full scan mode; 45–70 V capillary exit voltage; setting of skimmer 1, 10; setting of skimmer 2, 7; RF amplitude, 500; offset 0.9; trap, 10; extract, 10; ionization pulse time, 2500 ms; ionization delay time, 0.001 s; excitation sweep pulse 1, 2 ms; excitation sweep attenuation, 1:2.16 dB. Acquisition of spectra was performed with the Bruker Daltonik software XMASS and corresponding programs for mass calculation, data calibration, and processing. Peptide samples were dissolved in a solution of 3% acetic acid in 50% methanol:water. MALDI-time of flight mass spectrometry was performed with a Bruker BiFlex-DE mass spectrometer equipped with a Scout MALDI source and video system, a nitrogen UV laser (337 nm), and a dual channel plate detector. Sample preparation was performed with 1 μl of a freshly prepared saturated solution of α-cyano-4-hydroxycinnamic acid in acetonitrile, 0.1% trifluoroacetic acid (2:1), which was mixed with 0.5 μl of the peptide solution (34Bauer S.H. Wiechers M.F. Bruns K. Przybylski M. Stuermer C.A. Anal. Biochem. 2001; 298: 25-31Crossref PubMed Scopus (19) Google Scholar). Spectra were recorded at an accelerating voltage of 25 kV and were averaged over 40 single laser shots. Upon treatment of isolated PGI2 synthase with PN no nitrated tryptic peptide(s) could be initially found although immunoprecipitation of the enzyme with a monoclonal NT antibody, as well as conventional acid hydrolysis of the nitrated enzyme, indicated the presence of nitrated tyrosine. In previous work with other P450 proteins a nitration with PN resulted in proteolytic peptides with characteristic absorbance at 365 nm, from which the position of the 3-NT could be identified (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google Scholar, 14Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar). Because the postulated mechanism (35Mehl M. Daiber A. Herold S. Shoun H. Ullrich V. Nitric Oxide. 1999; 3: 142-152Crossref PubMed Scopus (98) Google Scholar, 36Zou M.H. Daiber A. Peterson J.A. Shoun H. Ullrich V. Arch. Biochem. Biophys. 2000; 376: 149-155Crossref PubMed Scopus (59) Google Scholar) suggested that only active PGI2 synthase can be nitrated, bovine aortic microsomes were first nitrated with increasing concentrations of PN and then the enzyme was isolated by gel electrophoresis (Fig.1). Western blot analyses shown in Fig. 1provided identical, specific bands at approximately 52 kDa up to a concentration of 500 μm PN that were stained by a polyclonal antibody against PGI2 synthase, and a monoclonal antibody against 3-NT, whereas higher concentrations than 500 μm caused unspecific additional staining of other proteins. The control also stained weakly, which probably was because of the presence of some atherosclerotic plaques in bovine arteries (37Zou M.H. Leist M. Ullrich V. Am. J. Pathol. 1999; 154: 1359-1365Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). A clear concentration dependence of PN on the extent of nitration was found up to 250 μm, which was at variance with the high affinity seen with the isolated enzyme (16Zou M.H. Ullrich V. FEBS Lett. 1996; 382: 101-104Crossref PubMed Scopus (198) Google Scholar, 17Zou M.H. Martin C. Ullrich V. Biol. Chem. 1997; 378: 707-713Crossref PubMed Scopus (270) Google Scholar), but may be explained by competitive targets for PN in the microsomal fraction. Indeed, when aortic microsomes were added to P450 BM-3 (Mr116,000) as a model protein for PGI2 synthase its Tyr-nitration was sharply decreased (Fig.2). Because the concentration of PGI2 synthase on the gel is very low its nitration hardly shows. If microsomes were treated with 5,5′-dithiobis(2-nitrobenzoic acid) to block SH groups their inhibitory effect is much less (data not shown), indicating that in microsomes protein thiols compete for PN and therefore higher PN concentrations are required as with the isolated enzyme. Previous studies with other P450 proteins and with model proteins (13Daiber A. Schöneich C. Schmidt P. Jung C. Ullrich V. J. Inorg. Biochem. 2000; 81: 213-220Crossref PubMed Scopus (35) Google Scholar,14Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, 38Jiao K. Mandapati S. Skipper P.L. Tannenbaum S.R. Wishnok J.S. Anal. Biochem. 2001; 293: 43-52Crossref PubMed Scopus (68) Google Scholar) had indicated that multiple Tyr nitrations may occur; therefore, in this study a PN concentration of 25 μm was selected that appeared most suitable to yield selective modification of a single Tyr residue. The inhibition of 6-keto-PGF1αformation was about 50 ± 15% compared with the inhibition at 250 μm, thus matching the NT staining intensities at 25versus 250 μm. Treatment and isolation of bovine aortic microsomes under these conditions (see "Experimental Procedures") provided ∼20 μg of PGI2 synthase isolated on SDS-PAGE from the 52-kDa band (Fig.3, lane D). The protein band was excised and subjected to proteolytic digestion using trypsin and mass spectrometric proteome analysis by MALDI-TOF as well as high resolution FT-ICR (39Fligge T.A. Reinhard C. Harter C. Wieland F.T. Przybylski M. Biochemistry. 2000; 39: 8491-8496Crossref PubMed Scopus (27) Google Scholar) (data not shown), which yielded unequivocal peptide fragment identification of the PGI2 synthase sequence. However, no NT-containing peptides or other modified peptide sites were detected by these mass spectrometric data (see TableI). With low abundance, ATP synthase (56 kDa) and peptide ions because of additional (unidentified) contaminating proteins in very low amounts were found by protein sequence data base analyses (SwissProt data base; data not shown). The contaminating proteins were estimated to account for maximally 20–30% of the protein band (see Fig. 3).Table IExpected cleavage pattern of PGI2 synthase after complete thermolysin digestionPossible Tyr-containing thermolysin fragment