Chemical Approach for Specific Enrichment and Mass Analysis of

Jul 17, 2009 - The analysis and detection of 3-nitrotyrosine are biologi- cally and clinically important because protein tyrosine nitration is known t...
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Anal. Chem. 2009, 81, 6620–6626

Chemical Approach for Specific Enrichment and Mass Analysis of Nitrated Peptides Jung Rok Lee,†,‡ Soo Jae Lee,§ Tae Woo Kim,†,‡ Jae Kyung Kim,†,‡ Hyung Soon Park,§ Dong-Eun Kim,| Kwang Pyo Kim,*,†,‡ and Woon-Seok Yeo*,| Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea, Institute of Biomedical Science and Technology, Konkuk University, Department of Molecular Biotechnology, Konkuk University, and Probiond Co., Ltd., Gwangjin VBS Center, Seoul 143-834, Korea The analysis and detection of 3-nitrotyrosine are biologically and clinically important because protein tyrosine nitration is known to be involved in a number of biological phenomena such as cellular signal transduction, pathogenesis of inflammatory responses, and age-related disorders. However, the main obstacles in the study are low abundance of nitrated species and lack of efficient enrichment methods. Here in, we suggest a new chemical approach to analyze nitrated peptides using mass spectrometry by incorporating specific tagging groups in the peptides through simple chemical transformations. Nitro groups on tyrosine side chains of nitrated peptides were subjected to reduction to give rise to amine which was further converted to metal-chelating motif. Mass analyses verified that Ni2+-NTA magnetic agarose beads selectively captured and isolated the modified peptides, i.e., nitrated peptides, by strong and specific metal chelating interactions. We further demonstrated the utility of our approach by detection of nitrated peptides in complex samples such as tryptic peptide mixtures of bovine serum albumin (BSA) and a HeLa cell lysate. Protein tyrosine nitration is a post-translational modification to substitute 3-positioned hydrogen to the nitro group in the phenol ring of tyrosine,1,2 mediated by oxidative and nitrosative stress such as peroxynitrite.3-7 This modification has been known to be associated with a number of diseases.8,9 For instance, cardiovascular diseases, including cardiovascular inflammation, * To whom correspondence should be addressed. E-mail: [email protected] (W.-S.Y.); [email protected] (K.P.K.). Fax: +82-2-2030-7890. † Institute of Biomedical Science and Technology, Konkuk University. ‡ Department of Molecular Biotechnology, Konkuk University. § Probiond Co., Ltd. | Department of Bioscience and Biotechnology, Konkuk University. (1) Ischiropoulos, H. Biochem. Biophys. Res. Commun. 2003, 305, 776–783. (2) Bartesaghi, S.; Ferrer-Sueta, G.; Peluffo, G.; Valez, V.; Zhang, H.; Kalyanaraman, B.; Radi, R. Amino Acids 2007, 32, 501–515. (3) Radi, R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4003–4008. (4) Reynolds, M. R.; Berry, R. W.; Binder, L. I. Biochemistry 2007, 46, 7325– 7336. (5) Ischiropoulos, H.; Beckman, J. S. J. Clin. Invest. 2003, 111, 163–169. (6) Lin, H. L.; Myshkin, E.; Waskell, L.; Hollenberg, P. F. Chem. Res. Toxicol. 2007, 20, 1612–1622. (7) Roberts, E. S.; Lin, H.; Crowley, J. R.; Vuletich, J. L.; Osawa, Y.; Hollenberg, P. F. Chem. Res. Toxicol. 1998, 11, 1067–1074. (8) Ischiropoulos, H. Arch. Biochem. Biophys. 1998, 356, 1–11. (9) Ryberg, H.; Caidahl, K. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2007, 851, 160–171.

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heart failure, and transplant coronary artery disease, elevate the amount of inducible nitric oxide synthase (iNOS) resulting in accumulation of tyrosine nitrated proteins.10-13 It was also reported that protein tyrosine nitration is associated with asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and acute respiratory distress syndrome (ARDS).14 3-Nitrotyrosine is relatively stable, therefore, has been regarded as an earliest marker for Alzheimer disease and amyotrophic lateral sclerosis (ALS).15-17 Recently, there have been efforts to search nitrated proteins in PC12 cells which terminally differentiate with treatment of nerve growth factor.18 Studies on the significance of protein tyrosine nitration in biological and clinical researches mentioned above necessitate effective methods to investigate the nitrated proteins. In practice, immunoassay with anti 3-nitrotyrosine antibodies is a widely used method and regarded as a useful technique for the observation of nitrated proteins.19,20 However, immunoassay cannot provide molecular-level understandings of tyrosine nitration in the specific proteins because of its limitation in determining precise locations of modifications. Recently, mass spectrometry has been introduced to identify the nitrated proteins. Especially, the tandem mass spectrometry showed the ability to determine nitration sites.21,22 Indeed, application of mass spectrometry, in combination with twodimensional polyacrylamide gel electrophoresis (PAGE) separation and Western blot analysis followed by in-gel digestion, to the investigation of protein tyrosine nitration has made it possible to (10) Turko, I. V.; Murad, F. Pharmacol. Rev. 2002, 54, 619–634. (11) Evans, T. J.; Buttery, L. D.; Carpenter, A.; Springall, D. R.; Polak, J. M.; Cohen, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9553–9558. (12) Tsikas, D.; Caidahl, K. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2005, 814, 1–9. (13) Peluffo, G.; Radi, R. Cardiovasc. Res. 2007, 75, 291–302. (14) Ricciardolo, F. L.; Di Stefano, A.; Sabatini, F.; Folkerts, G. Eur. J. Pharmacol. 2006, 533, 240–252. (15) Butterfield, D. A.; Reed, T. T.; Perluigi, M.; De Marco, C.; Coccia, R.; Keller, J. N.; Markesbery, W. R.; Sultana, R. Brain Res. 2007, 1148, 243–248. (16) Haussinger, D.; Schliess, F. Neurochem. Int. 2005, 47, 64–70. (17) Sacksteder, C. A.; Qian, W. J.; Knyushko, T. V.; Wang, H.; Chin, M. H.; Lacan, G.; Melega, W. P.; Camp, D. G., 2nd; Smith, R. D.; Smith, D. J.; Squier, T. C.; Bigelow, D. J. Biochemistry 2006, 45, 8009–8022. (18) Tedeschi, G.; Cappelletti, G.; Negri, A.; Pagliato, L.; Maggioni, M. G.; Maci, R.; Ronchi, S. Proteomics 2005, 5, 2422–2432. (19) Dremina, E. S.; Sharov, V. S.; Schoneich, C. J. Neurochem. 2005, 93, 1262– 1271. (20) Soderling, A. S.; Hultman, L.; Delbro, D.; Hojrup, P.; Caidahl, K. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2007, 851, 277–286. (21) Turko, I. V.; Murad, F. Methods Enzymol. 2005, 396, 266–275. (22) Park, S. W.; Huq, M. D.; Hu, X.; Wei, L. N. Mol. Cell. Proteomics 2005, 4, 300–309. 10.1021/ac9005099 CCC: $40.75  2009 American Chemical Society Published on Web 07/17/2009

Figure 1. Schematic presentation of specific tagging and enrichment strategy for mass analysis of low-abundance peptides. Only a subset of peptides in a crude mixture undergoes chemical reactions to afford reactive functional groups, which are subjected to specific tagging. The tagged peptides are then extracted and isolated to solid supports and subsequently analyzed by mass spectrometry, which gives highly enhanced signal corresponding to analyte peptides.

successfully identify a number of nitrated proteins/peptides.18,23 While the use of mass spectrometry is advantageous over immunoassay in terms of sensitivity and site-determining capability, nevertheless, proteome-wide analysis of nitrated proteins/ peptides is still challenging due to ultralow abundance of nitrotyrosine. In this regard, the method to enrich nitrated proteins/ peptides prior to subsequent mass analysis has strongly been demanded. A number of enrichment methods have been developed for phosphorylated or glycosylated proteins,24-33 however, very few effective enrichment methods for nitrated peptides have been reported, probably because of poor chemical reactivity of the nitro group on the tyrosine side chain.34 In this context, one of the alternatives would be a detour by converting the nitro group to the amine group which is chemically reactive and, therefore, can be used as a chemical handle to employ tagging groups in nitrated residues. As depicted in Figure 1, a subset of peptides in proteolytic mixture undergoes chemical reactions to afford reactive functional groups, i.e., amine in the case of nitrotyrosine. The resulting functional groups are modified with tagging groups through specific chemical reactions. The tagged peptides are then extracted and isolated to solid supports and subsequently analyzed by mass spectrometry which gives highly enhanced signal corresponding to the analyte peptide. As early examples, Nikov et al. reported a method which involved reduction of nitrotyrosine to corresponding aminotyrosine, selective anchoring of cleavable biotin tags, and specific enrichment of biotinylated peptides with (23) Koeck, T.; Levison, B.; Hazen, S. L.; Crabb, J. W.; Stuehr, D. J.; Aulak, K. S. Mol. Cell. Proteomics 2004, 3, 548–557. (24) Reinders, J.; Sickmann, A. Proteomics 2005, 5, 4052–4061. (25) Jensen, O. N. Curr. Opin. Chem. Biol. 2004, 8, 33–41. (26) Leitner, A.; Lindner, W. Proteomics 2006, 6, 5418–5434. (27) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. Anal. Chem. 2004, 76, 3935–3943. (28) Kweon, H. K.; Hakansson, K. Anal. Chem. 2006, 78, 1743–1749. (29) Zhou, H.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375–378. (30) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379–382. (31) Zhang, H.; Li, X. J.; Martin, D. B.; Aebersold, R. Nat. Biotechnol. 2003, 21, 660–666. (32) Kaji, H.; Saito, H.; Yamauchi, Y.; Shinkawa, T.; Taoka, M.; Hirabayashi, J.; Kasai, K.; Takahashi, N.; Isobe, T. Nat. Biotechnol. 2003, 21, 667–672. (33) Lewandrowski, U.; Moebius, J.; Walter, U.; Sickmann, A. Mol. Cell. Proteomics 2006, 5, 226–233. (34) Yeo, W. -S.; Lee, S. J.; Lee, J. R.; Kim, K. P. BMB Rep. 2008, 41, 194– 203.

streptavidin affinity chromatography,35 while Zhang et al. introduced an improved enrichment strategy by hooking up a thiol group, which showed better chemical reactivity than a nitro group, to nitrotyrosine.36 In the present study, we used a new chemical approach for the efficient enrichment and mass analysis of nitrated peptides. Our strategy utilized specific and strong interactions between positively charged metal ions bound on magnetic beads via nitrilotriacetic acid (NTA) and chelating groups incorporated onto nitrated peptides through chemical modifications.37 Fidelity of the approach was validated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), elucidating chemical conversions and specific isolation of analyte peptides. EXPERIMENTAL SECTION Materials. Human angiotensin II (Ang II, H2N-DRVYIHPFCOOH), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium dithionite (Na2S2O4), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), trizma base, tetranitromethane, pyridine-2-carboxaldehyde, sodium cyanoborohydride (Na(CN)BH3), ethylene diamine tetraacetic acid (EDTA), imidazole, ammonium bicarbonate, iodoacetamide, 2-(N-morpholino)ethanesulfonicacid(MES),NP-40,phenylmethanesulphonylfluoride (PMSF), sodium dodecyl sulfate (SDS), sodium chloride (NaCl), sodium deoxcholate, and dithiothreitol (DTT) were obtained from Sigma-Aldrich (St. Louis, MO). Sulfo-NHSacetate and bovine serum albumin (BSA) were purchased from Pierce Biotech (Rockford, IL). HPLC grade H2O and acetonitrile were from Burdick & Jackson (Muskegon, MI). The ziptip C18 pipet tip was from Millipore (MA). Nitrotyrosine containing angiotensin II (nitro-Ang II) was synthesized by Anygen (Gwang-ju, Korea), and sequencing grade trypsin was from Promega (Madison, WI). Complete mini protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). The micro BCA protein assay kit was from Thermo Scientific (35) Nikov, G.; Bhat, V.; Wishnok, J. S.; Tannenbaum, S. R. Anal. Biochem. 2003, 320, 214–222. (36) Zhang, Q.; Qian, W. J.; Knyushko, T. V.; Clauss, T. R.; Purvine, S. O.; Moore, R. J.; Sacksteder, C. A.; Chin, M. H.; Smith, D. J.; Camp, D. G., 2nd; Bigelow, D. J.; Smith, R. D. J. Proteome Res. 2007, 6, 2257–2268. (37) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598– 599.

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(Rockford), and R-cyano-4-hydroxycinnamic acid (HCCA) was obtained from Bruker Daltonics (Leipzig, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from WelGENE Inc. (Daegu, Korea). Trifluoroacetic acid (TFA) was purchased from Applied Biosystems (Foster City, CA), and Ni2+-NTA magnetic agarose beads were from Qiagen (Hilden, Germany). In Vitro Nitration of BSA. For in vitro nitration, bovine serum albumin (BSA) was incubated with 11 mM tetranitromethane (TNM) for 1 h at 37 °C in 5 mM Tris-HCl (pH 8.7). Lysis of HeLa Cells. HeLa cells were maintained in media containing Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified environment in 5% CO2. The cells were scraped from the dishes and then vortexed in RIPA buffer (150 mM NaCl, 0.1% SDS (w/v), 0.5% sodium deoxcholate (w/v), 1% NP-40 (w/v), 1 mM PMSF, 50 mM Tris-HCl (pH 8.0) and protease inhibitor cocktail (1×)) for 30 min at 4 °C. The cell lysate was centrifuged at 15 000g for 15 min at 4 °C, and the supernatant was removed. The protein concentration of the cleared lysate was quantified using the BCA assay kit. Tryptic Digestion of BSA, Nitrated-BSA, and HeLa Cell Lysate. BSA and in vitro nitrated BSA were dissolved in 50 µL of 50 mM ammonium bicarbonate (pH 7.8) and heated to boiling for 5 min. After the solutions were cooled to room temperature, the protein solutions were treated with aqueous dithiothreitol (DTT) to the final concentration of 5 mM and incubated for 30 min at 60 °C. Cysteine residues were then alkylated by incubation with 15 mM iodoacetamide for 30 min in the darkness, and then the protein solutions were digested overnight with trypsin at 37 °C. Tryptic in-gel digestion of HeLa cell lysate was performed as described previously.38 The digested peptides were desalted with a ziptip. Acetylation of Peptides and Reduction of Nitrotyrosine. For the acetylation of N-terminal amines and ε-amines of lysine residues, peptides were dissolved in 20 mM HEPES buffer (pH 7.8) and acetylated with sulfo-NHS-acetate (15-fold molar excess). The reaction mixture was incubated for 30 min at room temperature, quenched by addition of 1 N NaOH until pH 14, and further incubated for 20 min. Subsequently, the mixture was acidified by 1 N HCl to pH 8 and then desalted with a ziptip, and the eluents were dried. For reduction of the nitro group on tyrosine to the amine, the acetylated nitro-Ang II and acetylated nitro-BSA tryptic peptides were dissolved in 20 mM HEPES buffer (pH 8.0) and treated with Na2S2O4 (500-fold molar excess) for 30 min. Aminated angiotensin II (amino-Ang II) and aminated BSA tryptic peptides were obtained via desalting and drying as described above. Schiff Base Formation and Reductive Amination. Schiff base formation was carried out in 10 mM MES buffer at pH 5.0. The aminated Ang II and aminated BSA tryptic peptides were treated with pyridine-2-carboxaldehyde (10 000-fold molar excess) and 10 mM Na(CN)BH3. The resulting mixture was incubated for 30 min at room temperature and applied to a C18 column (38) Lee, S. J.; Lee, J. R.; Kim, Y. H.; Park, Y. S.; Park, S. I.; Park, H. S.; Kim, K. P. Rapid Commun. Mass Spectrom. 2007, 21, 2797–2804.

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(ziptip). The eluent was then dried by speedvac to give the bispyridinylated form of aminated peptides. Enrichment of Bispyridinylated Peptides. Bispyridinylated peptides were captured by Ni2+-NTA magnetic agarose beads. The beads were prewashed with 10 mM imidazole (pH 8.0) and placed in the tube on a magnetic separator, and the supernatant was removed after 1 min. The peptides which were reconstituted with 10 mM imidazole (pH 8.0) were incubated with Ni2+-NTA beads for 30 min. The tube was then placed on a magnetic separator, and the supernatant was removed after 1 min. To remove nonspecifically bound nontagged peptides, the beads were washed with 10 mM imidazole (pH 8.0). The bound bispyridinylated peptides on the beads were released with 300 mM imidazole or 10 mM EDTA (pH 8.0). The collected supernatant was desalted on a ziptip and analyzed by MALDI-TOF MS. Mass Spectrometry and Data Analysis. MALDI-TOF MS was performed with Autoflex III MALDI-TOF/TOF mass spectrometry (Bruker Daltonics, Germany) using a Smartbeam laser as an ionization source and R-cyano-4-hydroxycinnamic acid (HCCA, 1 mg/mL in 30% acetonitrile in water (v/v) containing 0.1% TFA) as a matrix. Typical experimental parameters included the following: reflector mode in positive ions, 19 kV accelerating voltage, and 50 Hz repetition rate. All spectra were obtained with an average of ∼1000 shots. For sample preparation, 1 µL of a peptide solution was mixed with 2 µL of freshly prepared matrix solution and 1 µL of the mixture was placed on the MALDI plate, which was allowed to dry at room temperature. To interpret the mass spectra obtained by MALDI, we used the MASCOT search engine (Matrix Science Inc., Boston, MA) using the National Center for Biotechnology Information database (NCBI, Bethesda, MD). The peptide mass tolerance was 200 ppm with one missed cleavage. We performed liquid chromatography (LC)-MS/MS analyses on an LTQ ion trap mass spectrometer (Thermo, San Jose, CA) equipped with an Ultimate nano HPLC (Dionex, Sunnyvale, CA) and nanospray source. The peptides were separated on a fusedsilica microcapillary C18 column (75 µm i.d., 100 mm length). LC was conducted under the followed linear gradient conditions: 0 min 3% B, 15 min 8% B, 23 min 16% B, 50 min 48% B, 52 min 90% B (buffer A, 0.1% formic acid in H2O; buffer B, 0.1% formic acid in acetonitrile). The column was eluted with 90% of solvent B for 10 min at the end of each run and then reequilibrated with the initial solvent condition, 3% solvent B, for the next run. The flow rate was 250 nL/min. The electrospray voltage was set at 2.0 kV, and the threshold for switching from MS to MS/MS was 500. The normalized collision energy for MS/MS was 35% of the main rf amplitude, and the duration of activation was 30 ms. All spectra were acquired in datadependent mode. Each full MS scan was followed by nine MS/ MS scans, corresponding from the most intense peak to the ninth intense peak of the full MS scan. The repeat count of the peak for dynamic exclusion was 1, and its repeat duration was 30 s. The dynamic exclusion duration was set to 180 s, and the exclusion mass width was ±1.5 Da. The list size of dynamic exclusion was 50. For database searching, TurboSEQUEST (Thermo Electron) was used with precursor and fragment ion mass tolerances of 1.5 and 1 Da, respectively.

Scheme 1. Chemical Synthetic Scheme to Convert the Nitro Group on a Tyrosine Side Chain to an Amino Group for Tethering Chelators Which Form a Stable Complex with Ni2+-NTA Magnetic Agarose Beads

Oxidation on Met (+16 Da), carboxyamidomethylation on Cys (+57 Da), niration on Tyr (+45 Da), and nitrosylation on Tyr (+29 Da) were selected as variable modifications. After a SEQUEST search with the combined peptide sequences, Xcorr must be over than 1.8, 2.2, and 3.3 for the +1, +2, and +3 charge states, respectively, with ∆Cn greater than 0.1. RESULTS AND DISCUSSION Experimental Design. As an enrichment strategy, we harness immobilized metal-ion affinity chromatography (IMAC) in which positively charged metal ions on solid supports specifically form a complex with a ligand of chemically modified peptide through chelation. The interaction between metal ions on solid supports and chelators on the peptides is not intrinsically covalent but, nevertheless, is strong enough to maintain the complex structure throughout the isolation steps. In addition, the captured peptides can easily be released from the solid support by salt, pH adjustment, or competing chelators. In this work, we utilize NTAmodified magnetic agarose beads as a solid support, Ni2+ as a metal ion, and imidazole and EDTA as competing chelators to release the peptides. Magnetic beads which were used herein to facilitate separation steps because the simple application of a magnetic field can effectively and physically separate this solid phase from the liquid phase. Our strategy starts with chemical conversion of the nitro group of the tyrosine side chain to an amine, which possesses high chemical reactivity so as to play a role of a chemical process to introduce the moieties of interest. Scheme 1 shows the chemical approach used in this study to capture 3-nitrotyrosine containing peptides. First, ε-amine of lysine and N-terminal amine are acetylated with sulfo-NHS-acetate to block the chemical reactivity of the amine. Next, the nitro group on a tyrosine is reduced to an amino group by treatment with sodium dithionite. The resulting amine is then reacted with pyridine-2-carboxaldehyde affording a Schiff base, i.e., imine which undergoes reductive amination upon treatment with Na(CN)BH3 to give rise to monopyridinylmethylamine. The second Schiff base formation ensues in the presence of a large excess of pyridine-2-carboxaldehyde, followed by another reductive amination, leading to the bispyridinylated peptide. We reason that the resulting bispyridinylated tyrosine would form a stable complex with Ni2+-NTA magnetic agarose beads. Although Scheme 1 depicts that two nitrogens in the pyridine rings chelate to Ni2+, we do not exclude

the possibility that the phenolic oxygen and the nitrogen at the 3-position could be involved in the complex formation as well. Mass Analysis of Chemical Conversions. We performed chemical reactions according to Scheme 1 and verified each step using MALDI TOF MS. First, Ang II and nitro-Ang II were mixed and analyzed with MALDI. The mass spectrum showed two distinctive peaks at m/z 1047.3 and m/z 1092.4, corresponding to Ang II and nitro-Ang II, respectively (Figure 2a). Next, the mixture in 20 mM HEPES buffer (pH 7.8) was treated with sulfo-NHSacetate, quenched by the addition of 1 N NaOH, acidified with 1 N HCl, and desalted. MS analysis of the mixture showed that the original peaks were absent but gave rise to two new peaks at m/z 1089.2 and 1134.2, corresponding to acetylated Ang II and nitroAng II, respectively (Figure 2b). We also observed the peaks at m/z 1076.4, m/z 1102.2, and 1118.2 (Figure 2a,b), corresponding to the photodecomposed species of nitrated peptides which lost

Figure 2. Mass analyses of chemical conversions: (a) The mass spectrum for the peptide mixture of Ang II (DRVYIHPF) and nitroAng II (DRVNO2YIHPF) displayed peaks at m/z 1047.3 and m/z 1092.4. (b) After acetylation with sulfo-NHS-acetate, the original peaks were not observed; however, there were new peaks at m/z 1089.2 (@DRVYIHPF) and m/z 1134.2 (@DRVNO2YIHPF) corresponding to acetylated products. (c) The reducing agent Na2S2O4 selectively reduced nitro-Ang II (m/z 1104.2, @DRVNH2YIHPF), whereas Ang II remained intact. (d) Schiff base formation and reductive amination in the presence of a large excess of pyridine-2-carboxaldehyde exclusively gave rise to the monopyridinylated and bispyridinylated peptide peaks at m/z 1195.9 (@DRVY#IHPF) and m/z 1287.0 (@DRVY*IHPF). [@, acetylation (mass shift of +42 Da) on N-terminal; #, monopyridinylation (mass shift of +106 Da) on tyrosine; *, bispyridinylation (mass shift of +197 Da) on tyrosine]. Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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one or two oxygens. These peaks are typical in nitrated tyrosine analysis using MALDI.38,39 In this study, the Smartbeam laser was used as an ionization source, which adopts Nd:YAG (Nd:Y3Al5O12). Although the emitting wavelength of YAG laser is different from that of the nitrogen laser, the characteristic photodecomposed peaks of nitrated peptides were observed. We have recently reported a distinctive peak pattern of nitrated peptides by using the Smartbeam.38 Upon treatment with Na2S2O4, the peak at m/z 1134.2 disappeared and a new peak at m/z 1104.2 appeared, while the peak at m/z 1089.2 was preserved (Figure 2c). The new peak at m/z 1104.2 was consistent with aminated product, amino-Ang II, thus verifying that Na2S2O4 selectively reduced nitro-Ang II to amino-Ang II, whereas Ang II remained intact. For the final step, the resulting mixture was treated with pyridine-2-carboxaldehyde and 10 mM Na(CN)BH3 in MES buffer at pH 5.0. The mass analysis gave the peak at m/z 1287.0, which corresponded to the bispyridinylated tyrosine containing peptides (Figure 2d). Again, this result showed that the second Schiff base formation ensued in the presence of an excess amount of pyridine-2-carboxaldehyde, followed by another reductive amination, leading to the bispyridinylated peptide. It should be noted that m/z 1195.9 in Figure 2d resulted from a trace amount of the monopyridinylated product. Indeed, as shown in Figure 2, the whole mass analyses for chemical conversions clearly indicated that the reactions proceeded in high yield and that our chemical conversion strategy was well suited for specific modification of a nitrotyrosine residue. Selective Isolation of Chemically Modified Peptides. Next, we tested our isolation strategy. We used the magnetic bead as a solid support because it can be effectively and easily separated from a liquid phase upon an exposure to a magnetic field facilitating separation steps in the experimental procedure. Thus, the peptide mixture of Ang II and bispyridinylated Ang II (Figure 3a) was reconstituted with 10 mM imidazole (pH 8.0) and incubated with Ni2+-NTA magnetic beads for 30 min. The beads were collected on a magnetic separator, and the supernatant was analyzed by MALDI-TOF MS. The mass spectrum showed one predominant peak at m/z 1089.7 corresponding to Ang II and a trace of bispyridinylated Ang II at m/z 1286.7 (Figure 3b). This result implied that the Ni2+-NTA beads selectively and efficiently captured bispyridinylated Ang II through specific interactions between Ni2+-NTA and bispyridinyl groups. The beads were then washed with 10 mM imidazole (pH 8.0), and the washing solution was analyzed. As revealed in Figure 3c, the predominant species in the washing solution was Ang II, indicating that the metal chelating interaction between Ni2+-NTA and bispyridinyl groups was strong enough to retain bound peptides during a washing step which removed nonspecifically bound nontagged peptides. Finally, the bound bispyridinylated Ang II on the beads was released with 300 mM imidazole or 10 mM EDTA (pH 8.0). Figure 3d shows the mass spectrum of an eluent that was obtained from imidazole elution, displaying the major peak at m/z 1286.7. This result implies that previously captured bispyridinylated Ang II can specifically be isolated, i.e., enriched for mass analysis by way of our strategy to harness a chemical approach. Intriguingly, we observed one more peak at (39) Petersson, A. S.; Steen, H.; Kalume, D. E.; Caidahl, K.; Roepstorff, P. J. Mass Spectrom. 2001, 36, 616–625.

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Figure 3. Selective isolation of the bispyridinylated Ang II using Ni2+NTA magnetic agarose beads. (a) The mass spectrum for the peptide mixture of Ang II and bispyridinylated Ang II as in Figure 2d. (b) Mass analysis of the nonbonding solution after incubation with Ni2+-NTA magnetic beads gave one major peak at m/z 1089.3, indicating selective capture of bispyridinylated Ang II on magnetic beads. (c) Washing eluent gave one major peak at m/z 1089.3 as well elucidating strong chelating interactions between Ni2+ and bispyridinyl cheating groups on peptides. (d) After release with imidazole, the mass spectrum displayed the major peak at m/z 1286.7, verifying the enrichment of the analyte peptides. Note that the peak at m/z 1342.7 corresponds to the Ni2+ adduct.

Figure 4. Enrichment of nitro-Ang II in BSA proteolytic mixture. (a) Mass analysis of the mixture showed distinctive peptide fragment peaks (sequences were presented) and nitro-Ang II (f). (b) After chemical modifications according to Scheme 1, the mixture was treated with Ni2+-NTA beads and the final eluent was analyzed by MALDI which gave only two distinctive peaks at m/z 1195.5 and m/z 1286.6, corresponding to monopyridinylated Ang II and bispyridinylated Ang II, respectively.

m/z 1342.7 in the imidazole eluent, which turned out to be the Ni2+ adduct of the analyte [M - H + Ni]+. EDTA eluent analysis gave the similar result; however, no Ni2+ adduct peak was observed because of complex formation between Ni2+ and EDTA (data not shown). Enrichment of Nitrated Peptides in BSA Proteolytic Mixture. We carried out a feasibility study on our strategy to enrich nitrated peptides using BSA (66 kDa) as a model system. Thus, 10 µg (approximately 150 pmol) of BSA was digested and mixed with 100 pmol of nitro-Ang II. Mass analysis of this mixture displayed typical peaks corresponding to BSA tryptic peptides and nitro-Ang II with similar intensities (Figure 4a). Two photodecomposed species of nitrated peptides were also observed. Next, the mixture underwent a series of chemical modifications involving acetylation, reduction, Schiff base formation, and reductive amination. Nitro-Ang II was converted into bispyridinylated Ang II (m/z 1286.6) with monopyridinylated Ang II (m/z 1195.5) as a

Figure 5. Isolation of nitro-AngII from HeLa cell lysate. Mass spectra that were obtained from spiked 100 pmol of nitro-AngII (a) and 100 fmol of nitro-AngII (b) showed the predominant peak at m/z 1286.7 corresponding to bispyridinylated Ang II.

minor reaction product, whereas other peptides were subjected only to acetylation including bisacetylation of lysine residues (data not shown), suggesting that our chemical modification scheme is specific to nitrotyrosine residues and is chemically orthogonal to other functional groups in the proteolytic mixture. Following the similar procedure as described above, the mixture was treated with Ni2+-NTA beads and the final eluent was analyzed. The mass spectra showed only two distinctive peaks at m/z 1195.5 and m/z 1286.6 corresponding to monopyridinylated Ang II and bispyridinylated Ang II, respectively (Figure 4b). Taken together, these results signify that the nitrated peptide, nitro-Ang II, can eventually be enriched for mass analysis, thus, clearly verifying the fidelity of our strategy. Isolation of Nitrated Angiotensin II from HeLa Cell Lysate. We next used cell lysate to confirm that our strategy is available to enrich the nitrated peptide in highly complex samples. Proteolytically digested peptides from HeLa cell lysate were prepared by trypsin in-gel digestion. A total of 100 pmol and 100 fmol of nitro-AngII were spiked into 10 µg of HeLa cell lysate, respectively. As described above, serial chemical reactions were performed with each sample such as acetylation, reduction, and Schiff base formation. The resulting samples were then treated with the Ni2+NTA bead. This procedure led to the detection of 100 pmol (Figure 5a) and 100 fmol of nitro-AngII (Figure 5b) from highly complex cell lysate. Identification of Nitrated BSA Tryptic Peptides in HeLa Cell Lysate. Finally, the proposed enrichment method was employed to enrich in vitro nitrated BSA tryptic peptides from HeLa cell lysate. BSA was in vitro nitrated with TNM and was digested in-solution with trypsin. The nitration sites of TNMtreated BSA were identified and confirmed with LC-MS/MS as shown previously.38 An aliquot of the HeLa cell lysate corresponding to 10 µg of the cellular proteins was separated on SDS-PAGE. The gel band region corresponding to the molecular weight of BSA was cut and subjected to in-gel tryptic digestion. To this resulting proteolytic mixture, 100 pmol and 100 fmol of tryptic peptides of nitrated BSA were spiked, respectively. The whole mixture was then subjected to a series of chemical conversions and separation steps. As shown in Figure 6, while nitrated BSA peptides were not detected in the resulting mixture before enrichment (Figure 6a), a total of six peaks from the 100 pmol sample (Figure 6b) and two peaks from the 100 fmol sample (Figure 6c) corresponding to the nitrated peptides (marked with f) of BSA were identified after isolation. The identified nitrated

Figure 6. Identification of in vitro nitrated BSA tryptic peptides in HeLa cell lysate. (a) The mass spectrum for the mixture of nitrated tryptic BSA peptides (100 pmol and 100 fmol) and 10 µg of tryptic peptides of HeLa cell lysate. The mixture was then subjected to a series of chemical conversions described in the text and treated with Ni2+-NTA beads. The final eluent was analyzed by MALDI, which gave a total of six peaks from the 100 pmol sample (part b) and two peaks from the 100 fmol sample (part c) corresponding to the nitrated peptides of BSA (f). Table 1. Summary of Nitrated BSA Peptides Identified after Enrichment Strategya sequence number

peptide sequence

MH+

157-167 161-167 337-346 341-346 360-371 375-386

K.FWGK@YLY*EIAR.R K.Y#LY#EIAR.R K.DVCKNY#[email protected] K.NY#[email protected] R.RHPEY#AVSVLLR.L K.EY#EATLEECCAK.D

1726.874 1181.610 1387.631 942.432 1587.875 1536.635

100 fmol of BSA

100 pmol of BSA

O O

O O O O O O

a O denotes that a nitration site is detected, and all of the N-terminals were acetylated. @, acetylation (mass shift of +42 Da) on lysine. #, monopyridinylation (mass shift of +106 Da) on tyrosine. *, bispyridinylation (mass shift of +197 Da) on tyrosine.

peptides of BSA are summarized in Table 1, clearly showing that most of the major peaks were assigned as nitrated peptides from nitrated BSA. For optimizing reaction conditions and experimental protocols, we have carried out multiple experiments. We have investigated the completeness of reactions including acetylation (Figure S1 in the Supporting Information), reduction (Figure S2 in the Supporting Information), and Schiff base formation (Figure S3 in the Supporting Information) and have characterized the reactions in time course with appropriate concentration of substrates (10 nmol, 100 pmol, 100 fmol). It turned out that the completeness of chemical conversions was seriously affected by concentrations of reagents not by reaction times. As shown in Figure 2, all of previous peaks disappeared in the next chemical conversion showing that the reactions in Scheme 1 proceeded with essentially quantitative yield by the procedure described in the Experimental Section. Finally, the analyses of nonbonding solutions and washing eluents gave no mono- and bispyridinylated peptides, which were converted from the nitro group on the nitrated tyrosine residue of Ang II. In addition, enrichment of nitrated Ang II in BSA Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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proteolytic mixture afforded exclusively two distinctive peaks corresponding to monopyridinylated Ang II and bispyridinylated Ang II with an enrichment factor of nearly 2.5 (Figure 4b). These results and Figure 5 confirmed that our strategy afforded the efficient affinity purification with high yield and purity. The existence of several hundreds of histidine-rich proteins in proteome raises a hampering issue in the affinity chromatography, which is not fully solved yet, although the 6XHis affinity purification is widely used for the specific enrichment and purification system for the 6XHis-tagged proteins in the current biological science field. In the current protocol, we modified the manufacturer’s recommending procedure by introducing more stringent washing procedure to minimize the nonspecific contaminants during enrichment of nitrated peptides with Ni2+-NTA magnetic beads (Figure S4 in the Supporting Information). To confirm the specificity of the enrichment method, the acquired MALDI spectra after enrichment of nitrated peptides from HeLa cell lysate (Figure 6b,c) were analyzed with peptide mass fingerprinting using MASCOT to check out whether histidinerich peptides were included in the enriched peptide preparation. MALDI analysis with peptide mass fingerprinting following the enrichment failed to detect such contaminating peptides. It suggests that our enrichment procedure includes a proper washing step to minimize such nonspecific contaminants. CONCLUSIONS AND PERSPECTIVES This report describes a new nitrotyrosine enrichment strategy harnessing a chemical approach which includes the conversion

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of a nitro group to an amine, derivatization of the amine to metal chelators, and solid phase capture/release on magnetic beads. By using the BSA proteolytic mixture as a model system, we demonstrated that our strategy provides a reliable and effective enrichment procedure for nitrated peptides. This method would be suitable for application to more complex systems. Finally, the nitrated peptide enrichment method described herein is expected to be a significant addition to analytical tools for protein posttranslational modifications that are central mechanisms to regulate the functions of cellular proteins. ACKNOWLEDGMENT This study was supported by grants from the 21C Frontier Functional Proteomics Project from the Korean Ministry of Science & Technology (Grant FPR08 A1-032), Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology (grant SC2120), and Industrial Technology Development, Ministry of Knowledge Economy (grant 10032113), Republic of Korea. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 9, 2009. Accepted July 6, 2009. AC9005099