Selective Enrichment and Mass Spectrometric Identification of

Bioinformatic analysis revealed that tyrosine nitration occurred within the specific functional domains of the two nitrated proteins (Table 2). Furthe...
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Anal. Chem. 2011, 83, 157–163

Selective Enrichment and Mass Spectrometric Identification of Nitrated Peptides Using Fluorinated Carbon Tags Jae Kyung Kim,†,‡ Jung Rok Lee,†,‡ Jeong Won Kang,† Soo Jae Lee,† Gu Choul Shin,§ Woon-Seok Yeo,| Kyun-Hwan Kim,‡,§ Hyung Soon Park,⊥ and Kwang Pyo Kim*,†,‡ Department of Molecular Biotechnology, WCU Program; Institute of Biomedical Science and Technology; Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine, Konkuk University School of Medicine; Department of Bioscience and Biotechnology; and Probiond Co., Ltd., Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Korea Protein tyrosine nitration (PTN) is a post-translational modification that is related to several acute or chronic diseases. PTN introduces a nitro group in the ortho position of the phenolic hydroxyl group of tyrosine residues. PTN has been shown to be involved in the pathogenesis of inflammatory responses, cancers, and neurodegenerative and age-related disorders. Furthermore, it has been proposed that PTN regulates signal cascades related to nitric oxide (NO · ) production and NO-mediated processes. Although nitrated proteins as markers of oxidative stress are confirmed by immunological assays in various affected cells or tissues, it is not known how many different types of proteins in living cells are nitrated. Since protein nitration is a low-abundance post-translational modification, development of an effective enrichment method for nitrated proteins is needed to detect nitrated peptides or proteins from the limited amount of pathophysiological samples. In the present study, we developed an enrichment method using specific chemical tagging. Nitroproteome profiling using chemical tagging and mass spectrometry was validated by model proteins. Furthermore, we successfully identified numerous nitrated proteins from the Huh7 human hepatoma cell line. Protein tyrosine nitration (PTN) is a post-translational modification process that adds a nitro group (NO2) in the ortho position of a tyrosine ring to yield 3-nitrotyrosine.1 This modification is mediated by the reaction of tyrosine with the nitrating species, peroxynitrite (ONOO-), which is generated from the reaction of nitric oxide (NO · ) and superoxide anion (O2 · -). As a result of protein tyrosine nitration, physical properties of nitrated proteins are changed. Nitrated proteins usually show a loss of function as observed in skeletal muscle * To whom correspondence should be addressed. E-mail: kpkim@konkuk. ac.kr. Fax: +82-2-450-3395. † Department of Molecular Biotechnology. ‡ Institute of Biomedical Science and Technology. § Department of Pharmacology and Center for Cancer Research and Diagnostic Medicine. | Department of Bioscience and Biotechnology. ⊥ Probiond Co. (1) Ischiropoulos, H. Biochem. Biophys. Res. Commun. 2003, 305, 776–783. 10.1021/ac102080d  2011 American Chemical Society Published on Web 12/13/2010

sarcoplasmic reticulum Ca-ATPase (SERCA),2 prostacyclin synthase,3 and ERK,4 as well as decreased solubility as observed in a-synuclein to form protein aggregates in Parkinson’s disease.5 Protein tyrosine nitration is emerging as a pathophysiological event involved in numerous biological processes as well as pathological events such as cytoskeletal dysfunction,6 platelet activation,7 stroke, acute respiratory disease, asthma, and neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS).8 However, little is known about the mechanisms and the target proteins involved in the modification because of the low stoichiometry of nitrotyrosines in proteins. The levels of nitrotyrosine have been reported as 10-100 pmol/mg or about 1-5 nitrated residues per 10 000 tyrosines.9 Therefore, it is necessary to develop new analytical methods to efficiently enrich and identify nitrated proteins from various biological samples. Most available methods are based on an immunochemical enrichment approach followed by mass spectrometric analysis.10,11 Despite their relevance, the lack of efficient antibodies to enrich the nitrated proteins has limited the expansion of the study of nitrated proteins at the proteome level. In the present study, we attempted to develop an enrichment method using chemical tagging of nitro groups on the modified tyrosines followed by affinity enrichment. We previously reported another method for the enrichment of nitrated peptides based on (2) Xu, S.; Ying, J.; Jiang, B.; Guo, W.; Adachi, T.; Sharov, V.; Lazar, H.; Menzoian, J.; Knyushko, T. V.; Bigelow, D.; Schoneich, C.; Cohen, R. A. Am. J. Physiol. Heart Circ. Physiol. 2006, 290, H2220–2227. (3) Schmidt, P.; Youhnovski, N.; Daiber, A.; Balan, A.; Arsic, M.; Bachschmid, M.; Przybylski, M.; Ullrich, V. J. Biol. Chem. 2003, 278, 12813–12819. (4) Pinzar, E.; Wang, T.; Garrido, M. R.; Xu, W.; Levy, P.; Bottari, S. P. FEBS. Lett. 2005, 579, 5100–5104. (5) Reynolds, M. R.; Berry, R. W.; Binder, L. I. Biochemistry 2005, 44, 1690– 1700. (6) Nonnis, S.; Cappelletti, G.; Taverna, F.; Ronchi, C.; Ronchi, S.; Negri, A.; Grassi, E.; Tedeschi, G. Neurochem. Res. 2008, 33, 518–525. (7) Sabetkar, M.; Low, S. Y.; Bradley, N. J.; Jacobs, M.; Naseem, K. M.; Richard Bruckdorfer, K. Platelets 2008, 19, 282–292. (8) Lee, J. R.; Kim, J. K.; Lee, S. J.; Kim, K. P. Arch. Pharm. Res. 2009, 32, 1109–1118. (9) Radi, R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4003–4008. (10) Yeo, W. S.; Lee, S. J.; Lee, J. R.; Kim, K. P. Biochem. Mol. Biol. Rep. 2008, 41, 194–203. (11) Zhan, X.; Desiderio, D. M. Methods Mol. Biol. 2009, 566, 137–163.

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incorporation of a metal chelating motif into the modified tyrosine residues. This strategy comprised a series of chemical conversions to convert the nitro groups on the tyrosine residues to the metal chelating groups followed by solid-phase extraction with Ni2+nitrilotriacetic acid (NTA) magnetic agarose beads.12 Here, we describe an efficient enrichment method of nitrated peptides/proteins based on fluorine-fluorine interaction affinity purification following chemical conversions of the nitro groups on tyrosine residues to highly fluorinated moieties. Fluorinated carbon groups prefer to be localized in a fluorine-rich environment because of their unique solvophobic properties. Fluorinated solidphase extraction (FSPE) utilized these distinctive properties to isolate highly fluorinated peptides from nonlabeled peptides.13 The developed enrichment method for nitrated peptides was successfully applied to in vivo model systems to validate the feasibility of the method. In the present study, we assessed 28 different nitrated proteins from the hepatoma cell (Huh7) lysate, following selective enrichment of nitrated peptides and LC-MS/MS analysis. EXPERIMENTAL SECTION Materials. Human angiotensin II (Ang II, H2N-DRVYIHPFCOOH), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium dithionite (Na2S2O4), ammonium formate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ammonium bicarbonate, iodoacetamide, sodium dodecyl sulfate (SDS), sodium chloride (NaCl), and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St. Louis, MO). Sulfo-NHS-acetate, bovine serum albumin (BSA), a Pepclean C18 spin column, a Micro BCA protein assay kit, and GelCode Blue Stain Reagent were obtained from Thermo Scientific (Rockford, IL). NSuccinimidyl 3-(perfluorobutyl)propionate and FluoroFlash silica gel were purchased from Fluorous Technologies (Pittsburgh, PA). HPLC grade H2O and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI). A Ziptip C18 pipet tip was obtained from Millipore (MA). A Sep-Pak C18 cartridge was purchased from Waters (Milford, MA). Nitrotyrosine containing angiotensin II (nitro-Ang II) was synthesized by Anygen (Gwang-ju, Korea), and sequencing grade trypsin was obtained from Promega (Madison, WI). Complete mini protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). R-Cyano-4-hydroxycinnamic acid (CHCA) was produced by 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 tetrahydrofuran (THF) was purchased from Junsei (Tokyo, Japan). A 4-12% gradient Novex Bis-tris gel was purchased from Invitrogen Corp. (Carlsbad, CA). Lysis of the Huh7 Cell Line and Quantification. Huh7 cells were grown in DMEM containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 °C under 5% CO2. The proteins were extracted from the cell lysates using RIPA buffer [150 mM NaCl, 0.1% SDS (w/v), 0.5% sodium deoxcho(12) Lee, J. R.; Lee, S. J.; Kim, T. W.; Kim, J. K.; Park, H. S.; Kim, D. E.; Kim, K. P.; Yeo, W. S. Anal. Chem. 2009, 81, 6620–6626. (13) Go, E. P.; Uritboonthai, W.; Apon, J. V.; Trauger, S. A.; Nordstrom, A.; O’Maille, G.; Brittain, S. M.; Peters, E. C.; Siuzdak, G. J. Proteome Res. 2007, 6, 1492–1499.

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late (w/v), 1% NP-40 (w/v), 1 mM PMSF, 50 mM Tris-HCl (pH 8.0), and protease inhibitor cocktail]. The cell lysate was incubated for 30 min at 4 °C and centrifuged at 15 000 g for 15 min at 4 °C, and the supernatant was removed. Protein quantification was performed using a BCA assay kit. Trypsin Digestion of BSA and Huh7 Cell Lysate. BSA and the Huh7 cell lysate were dissolved in 50 µL of 50 mM ammonium bicarbonate (pH 7.8) and then boiled for 5 min to denature the proteins. Protein samples (total 500 µg) from the cell lysate were loaded on 4-12% gradient Novex Bis-tris gel shortly, and total proteins were divided into several wells. After loading, the gel was stained with the GelCode Blue Stain Reagent. The gel lanes were cut to the same size. Subsequently, reduction steps were performed by adding dithiothreitol (DTT) to give a final concentration of 5 mM in water and then incubating the samples for 30 min at 60 °C. Next, reduced cysteine residues were alkylated to block rebinding of thiol groups by incubating the samples with 50 mM iodoacetamide in darkness for 30 min at room temperature. The resulting cell lysates were then digested using trypsin for 14 h at 37 °C. The digested peptides were desalted using a Pepclean C18 spin column and a Sep-pak. Acetylation and Reduction Steps. Our method requires the attachment of fluorinated carbon tags to enrich nitrated peptides. Because fluorinated carbon tags react with amine groups, an acetylation step is required to prevent nonspecific attachment with N-terminal amines and ε-amines of lysine residues. Tryptic peptides were dissolved in 100 µL of 100 mM HEPES (pH 8.5), and sulfo-NHS-acetate (15-fold molar excess) was added to the peptides, which were incubated for 30 min at room temperature. The reaction was quenched by NaOH and HCl, and the reaction mixture was desalted using Ziptip and evaporated. The resulting acetylated peptides were dissolved in 100 µL of 100 mM HEPES (pH 8.5), reduced by sodium dithionite (500-fold molar excess) for 30 min at room temperature, desalted using Ziptip, and evaporated. Fluorinated Carbon Tagging Step. The reduced peptides were dissolved in 50 µL of 250 mM sodium bicarbonate (pH 8.5). The reagent solution was prepared by dissolving 250 mM N-succinimidyl 3-(perfluorobutyl)propionate in acetonitrile and mixed with the peptide solution. The reaction was allowed to proceed for 2 h at room temperature, followed by drying and washing the remaining reagent with tetrahydrofuran (THF). FSPE Step. Fluorinated carbon-tagged peptides were captured by fluorinated carbon-linked silica beads. Briefly, 5 mg of fluorinated carbon-linked silica beads with a diameter of 40 µm was put in a spin column. The beads were then prewashed with 200 µL of 80% methanol in 10 mM ammonium formate and subsequently centrifuged at 1500g for 1 min. To equilibrate the beads, 30% methanol in 10 mM ammonium formate was added to the tubes, and the samples were then centrifuged under the same conditions as described above. The fluorinated carbon-tagged peptides were dissolved in 200 µL of 30% methanol in 10 mM ammonium formate. The binding step was the same as the equilibrium step. Nonspecifically bound peptides were removed by washing with 30% methanol in 10 mM ammonium formate several times. Finally, the samples were eluted using 100% methanol as the elution buffer, and then the eluates were dried.

Scheme 1. The Enrichment Method Was Composed of Four Steps Including Modifications of Specific Residues, Tagging, and FSPE

Figure 1. Confirmation of chemical conversions. (a) Before chemical reaction, MALDI mass spectra of the peptide mixture of Ang II and nitroAng II indicated peaks at m/z 1046.3 and m/z 1092.4. (b) After the acetylation reaction, all displayed peaks were shifted to +42 Da and appeared at m/z 1088.1 and m/z 1134.2. (c) Sodium hydrosulfite reduced nitro-Ang II to amino-AngII displayed m/z 1104.2, while Ang II remained acetylated. (d) The last step of the chemical conversion was attachment of a fluorinated carbon tag to the amino group. N-Succinimidyl 3-(perfluorobutyl)propionate reacted only with amino-AngII and produced fluorinated carbon-tagged AngII, displayed at m/z 1379.0.

MALDI-TOF/TOF Analysis. MALDI-TOF/TOF experiments were conducted with an AutoFlex III (Bruker Daltonics, Germany) using a Smartbeam laser (Nd:YAG, 355 nm, 100 Hz) in positive ion reflector mode. The accelerating voltage was set to 19 kV, and all spectra were obtained by averaging at least 1000 laser shots. In addition, samples were mixed with R-cyano-4-hydroxycinnamic acid matrix solution (CHCA, 1 mg/mL in water containing 30% ACN and 0.1% TFA), after which mixtures of 1 µL of sample and matrix were spotted on the target and dried at room temperature. LC-ESI-IT-MS/MS Analysis. Nitrated peptides in Huh7 cell lysate were analyzed by LC-ESI-IT-MS/MS on an LTQ (Thermo, San Jose, CA) equipped with a nano-ESI source in positive ion mode. The reverse phase capillaries were prepared in the laboratory by adding 5 µm C18 particles into 75 µm × 12 fused silica capillaries. Two solvents were used to prepare the mobile phase, A and B. Solvent A was made of 0.1% formic acid in water, and solvent B was made of 0.1% formic acid in ACN. The gradient used for separation was solvent B (3-60% over 35 min; 60-100% over 45 min) over 60 min at flow rate of 0.2 µL/min. An LTQ instrument was operated continuously in data-dependent mode using 35% collision energy during separation. Database Searches and Validations. TurboSEQUEST (Thermo Electron) was used to identify the enriched peptides by searching tandem MS spectra in a NCBI human reference database that contained a forward sequence database (having 38 871 human protein sequence entries as of February 3, 2009). The search parameters for the trypsin enzyme: precursor and fragment tolerance, ±1.0 and ±0.5 m/z; number of missed cleavage sites, 2. Each modification involved oxidation of methionine (+16 Da), carbamidomethylation of cysteine (+57 Da), acetylation of lysine and N-terminal aminos (+42 Da), and fluorinated carbon taaged tyrosine (+289 Da). All MS/MS spectra of nitrated peptides were confirmed by manual validations. Gene Ontology (GO) Analysis. Localization information was obtained from the Protein Information Resource (http://pir. georgetown.edu/). To identify the molecular functions and biological pathways of the enriched proteins, GI numbers of NCBI were converted to Entrez gene ID numbers in the Protein Information Resource (PIR) Web site. Information regarding the molecular functions and biological pathways was also obtained using the PANTHER Web site (http://www.pantherdb.org/).

with nitrotyrosine residues. This strategy involves several chemical modification steps including acetylation of ε-NH2 groups on lysine residues and the N-terminus, reduction of NO2 groups on the nitrated tyrosine residues to NH2 groups, and fluorinated carbon attachment of the derivatized NH2 groups on the tyrosine residues. We confirmed each step in the chemical conversion using a peptide mixture of the equimolar amount of AngII and nitro-AngII (AngII; m/z 1046.3, nitro-AngII; m/z 1092.4) with MALDI TOF (Figure 1a). To prevent side reactions during the fluorinated carbon tagging step with N-succinimidyl 3-(perfluorobutyl)propionate, NH2 groups at the N-terminus and ε-NH2 groups of lysine residues were blocked by acetylation. After the peptides mixture was acetylated with an excess of sulfo-NHS-acetate, the peaks corresponding to the acetylated peptides showed a 42 Da increase in molecular weight and appeared at m/z 1088.1 and m/z 1134.2 (Figure 1b), which suggested that the acetylation was complete. The mass spectrum of the acetylated peptide mixture revealed that the two versions of Ang II were singly acetylated, resulting in a mass increase of 42 Da. We also detected peaks at m/z 1118.2 and m/z 1102.2, which corresponded to the photodecomposed nitro-Ang II with nitrosotyrosine and nitrenetyrosine, respectively (Figure 1a,b).14 Next, reduction was conducted, in which NO2 groups on the nitrotyrosine were reduced to NH2 groups via reaction with sodium hydrosulfite.15 The resulting NH2 group was then reacted with N-succinimidyl 3-(perfluorobutyl)propionate to generate perfluoralkyl derivatized peptide, which carries the affinity tag for the FSPE step. Because AngII peptides do not have NO2 groups, the m/z of acetylated AngII peptides did not show a mass shift after reduction. Peaks corresponding to nitroAngII peptide shifted from m/z 1134.2 to m/z 1104.2 (Figure

RESULTS AND DISCUSSION Confirmation of Chemical Conversions. Scheme 1 describes the entire chemical reaction process required to enrich peptides

(14) 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. (15) Sokolovsky, M.; Riordan, J. F.; Vallee, B. L. Biochem. Biophys. Res. Commun. 1967, 27, 20–25.

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1c) as a result of the reduction. The peak intensity of the aminated product, amino-AngII, decreased significantly upon reduction of the NO2 group. The reaction was shown to be very efficient since singly acetylated and aminotyrosine-containing Ang II was readily detected, whereas nitrated AngII was barely detectable after reduction of nitrotyrosine (Figure 1c). The resulting amino-AngII bearing a chemical functionality for fluorinated carbon tagging was selectively labeled with perfluoroalkyl groups using a chemoselective reaction. The NH2 group converted from the NO2 group of nitro-AngII participated in a reaction with N-succinimidyl 3-(perfluorobutyl)propionate to generate fluorinated carbon-tagged AngII by forming an amide bond. MALDI MS analysis of the final step gave a peak at m/z 1379.0, which corresponded to the fluorinated carbon-tagged AngII peptide (Figure 1d). Overall, the chemical conversions introducing an affinity tag into the nitro group on the nitrated tyrosine residues appeared to be complete, as indicated by the disappearance of the initial peak at m/z 1092.4 corresponding to the nitro-AngII and the appearance of a peak at m/z 1379 corresponding to the fluorinated carbontagged AngII, the final chemical conversion product. Separation of Fluorinated Carbon-Tagged Peptides. The FSPE is based on a fluorine-fluorine interaction.16 Because perfluoroalkyl groups are known to be ultrahydrophobic, FSPE occurs with high affinity and selectivity through dipole-dipole interactions among perfluoroalkyl groups. To separate nitrated peptides that were converted to carry perfluoroalkyl groups, we used fluorinated carbon-linked silica beads, which have affinities with fluorine groups in the affinity tag. The resulting peptide mixtures of acetylated Ang II (m/z 1088.6) and fluorinated carbontagged Ang II (m/z 1379.0) were reconstituted with 10 mM ammonium formate containing 35% methanol. In this binding buffer, the interactions were kept intact between fluorinated carbon-tagged peptides and beads, and these tagged peptides were captured by the beads. Because peptides which had no perfluoroalkyl group do not have affinity for the beads, the peptides indicated by m/z 1088.7 passed through the spin column (Figure 2b). MS analysis showed that peaks corresponding to the acetylated peptides as well as sodium (+22 Da) and potassium (+38 Da) adducts were detected (Figure 2b). As shown in Figure 2c, the acetylated Ang II peptides, which had no fluorine groups, were removed by washing with 35% methanol in 10 mM ammonium formate solution. Interaction between fluorinated carbonlinked silica beads and the fluorinated carbon tags was strong enough to keep the tagged peptides bound during the washing step. Finally, we were able to elute the fluorinated carbon-tagged peptides using 100% methanol with minimal contaminants. Figure 2d shows the mass spectrum of the eluent that has a major peak corresponding to the fluorinated carbon-tagged AngII, as indicated by a m/z value of 1379.0. These results demonstrated that the developed method could separate peptides that had fluorinated carbon tags with high selectivity. Enrichment of Nitropeptides in BSA Tryptic Peptide Mixture. As previously shown, the protocol (Scheme 1) was proven to have enough selectivity with high efficiency for enrichment of the nitrated peptide from the peptide mixture with the same molar (16) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nat. Biotechnol. 2005, 23, 463–468.

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Figure 2. Separation of affinity-tagged peptides. (a) The MALDI mass spectrum of the two peptides of acetylated Ang II and fluorinated carbon-tagged Ang II shown in Figure 1d. (b) Mass analysis of the nonbonding solution through a spin column filled with fluorinated carbon-linked silica beads showed a peptide peak at m/z 1088.7, while no fluorinated carbon tag containing peak was found. (c) The peptides indicated by m/z 1088.7 were washed with washing solution. (d) The remaining fluorinated carbon-tagged peptides were released by 100% MeOH and showed a peak at m/z 1379.0.

Figure 3. Enrichment of nitropeptide in BSA tryptic peptide mixture. (a) MALDI mass analysis of the mixture of nitro-AngII (f) and BSA spiked with peptides. (b) Following chemical conversion and FSPE according to Scheme 1, the final eluent was analyzed by MALDI, which gave just one peak at m/z 1378.6.

amount of nitrated peptide and the corresponding nonmodified peptide. To validate our enrichment method, we applied a more complex sample as a model system. Specifically, 100 pmol of nitro AngII peptides was spiked into a matrix of 10 µg of trypsin-digested of BSA. MALDI MS analysis of the BSA peptide mixture showed that 100 pmol of nitrated peptide generated a peak with very low intensity, marked with a star (Figure 3a). After enrichment via a series of chemical conversions, all BSA peptides were removed and the intensity of the originally nitrated peptide was prominent (Figure 3b). These findings clearly demonstrate that our enrichment method can be applicable to samples with high complexity such as total cell lysates or biological fluids. Identification of Endogeneous Nitroproteins from Huh7 Cell Lysate. To validate the enrichment method using a model biological system, the developed method was applied to enrich the nitrated peptides from total cell lysates of the Huh7 cell line. Because the nitration process is believed to be related to various pathological events, including cancers, nitrated proteins can potentially be used as biomarkers of various diseases. The tryptic peptide mixture of Huh7 cell lysate was prepared by in-gel tryptic digestion following SDS-PAGE separation of total cell lysate

Table 1. Summary of Identified Huh7 Peptides Using Fluorinated Carbon Tagging and FSPE. ]: N-Terminal Acetylation, ∧: Lysine Acetylation (mass shift of +42 Da), #: Methionine Oxidation (mass shift of +16 Da), *: Cysteine Alkylation (mass shift of +57 Da), @: Tyrosine Fluorinated Carbon Tagging (mass shift of +289 Da) reference

MH+

z

accession

3121 2373 3277 3078 1690 2714 2242 2602 1876 1509 1845 3399 2063 2282 1432

3 2 3 3 2 3 3 3 3 2 3 3 2 2 2

peptide ˆ .V R.T]GHKGY@VAFLESLELYYPQLYKK ˆ .L K.P]FSLTKDVLLDVAYAY@GK K.C]TCDETGYSGATC*HNSIYEPSC*[email protected] ˆ .I K.I]AC*LAGIVFILSGLCSMTGCSLY@ANK ˆ .D R.M]NEGDAYY@LLK R.M][email protected] ˆ .L K.L]RY@TGPEDADYTNLIK K.V]PANY@FHIC*SAILM#GFSLSK.S ˆ VLK.W K.P]SY@SDIAANPK ˆ .K R.V]PDRIMNY@K K.E][email protected] R.L]LDEY@NVTPSPPGTVLTSALSPVIC*GPNR.S -.M][email protected] R.N][email protected] R.L][email protected]

XC

caspase recruitment domain protein 9 isoform 1 cell cycle progression 2 protein isoform 1 cell recognition molecule Caspr2 precursor claudin 10 isoform a DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 deiodinase, iodothyronine, type II isoform b FRAS1-related extracellular matrix protein 2 precursor hypothetical protein LOC150590 hypothetical protein LOC51306 isoform 3 hypothetical protein LOC56985 karyopherin beta 1 notch 2 preproprotein PREDICTED: similar to hCG1728885 PREDICTED: similar to hCG2002956, partial protein phosphatase 1, regulatory (inhibitor) subunit 12A isoform b ralA binding protein 1 regulator of nonsense transcripts 1 ring finger protein 166 RYK receptor-like tyrosine kinase isoform 1 sciellin isoform a solute carrier family 32, member 1 stabilin 2 precursor structural maintenance of chromosomes 2 tastin isoform 1 transmembrane protein 62 transmembrane protein 87A isoform 1 zinc finger and BTB domain containing 5 zinc finger protein 30 isoform a

2.342 2.660 2.348 2.447 1.934 2.783 2.409 2.671 2.087 1.537 2.505 2.337 2.775 1.581 2.228

47717131 40217812 7662350 33598916 29725619 55749998 79749430 21389621 156142192 94158594 19923142 24041035 169161132 169217458 219842214

1768 2156 2314 2420 2234 1115 2108 1647 1651 2972 1437 1470 2103

2 2 3 3 2 2 2 2 3 3 2 2 2

ˆ [email protected] K.S]K K.L][email protected] R.V]VC*PIC*SAM#[email protected] R.A][email protected] K.Q][email protected] K.F][email protected] R.I][email protected] ˆ .I K.R]RY@TIIPLNK R.L][email protected] K.G]NHDAFNIPSLDSIKNY@[email protected] R.N][email protected] K.P][email protected] ˆ ECGK ˆ AFISR.H K.P]Y@EC*K

2.046 2.219 2.693 2.206 2.101 2.730 1.512 2.234 2.492 2.514 1.890 1.607 2.318

5803145 18375673 30520320 54607020 21536306 17999520 61743980 110347425 154800453 52851437 31377765 7662074 152963635

corresponding to 500 µg of cellular proteome. The tryptic peptides were subject to affinity enrichment with FSPE following a series of chemical conversions. LC-ESI-MS/MS analysis followed by FSPE clearly identified 28 nitrated peptides from the same numbers of proteins as summarized in Table 1. This result was deduced from triplicate experiments and peptides derived from 500 µg of Huh7 cell lysate were used in each experiment. The MS/MS spectra of the resulting 28 nitrated peptides were validated manually. The identification of the fluorinated carbontagged peptides derived from the nitrated peptides was confirmed by MS/MS. The nitropeptides were considered positive when their amine groups on the N-terminus and lysine residues were properly acetylated. Figure 4 shows representative MS/MS spectra of the fluorinated carbon-tagged peptides enriched with FSPE. These results indicated that the acetylation and subsequent reduction effectively blocked the nonspecific affinity tagging and selectively introduced the perfluoroalkyl groups serving as affinity tags for further enrichment of the nitrated peptides. Bioinformatic Analysis of Enriched Nitro Proteins. We found that a total of 28 nitrated peptides formed in the same numbers of proteins using fluorinated carbon tagging based enrichment of nitrated peptides followed by LC-ESI-MS/MS. We classified the identified proteins based on their subcellular localization, molecular function, and biological process according to gene ontology (GO) annotation using the HPRD (http:// www.hprd.org) and PANTHER (http://www.pantherdb.org) databases. Gene ontology annotation suggested that the nitroproteins are primarily localized in the plasma membrane (36%), nucleus (14%), and cytoplasm (11%), as summarized in Figure S1 in the Supporting Information. As shown in Figure 5a, the major molecular functions of the identified proteins were nucleic acid binding (11%), enzymes such as hydrolase (17%) and kinase (7%),

transcription factors (7%), receptors (7%), and signaling molecules (7%). GO analysis by the biological processes revealed that nitrated proteins are related to biological processes such as signal transduction (14%), developmental processes (11%), and the cell cycle (12%), as summarized in Figure 5b. To elucidate the biological effects of protein tyrosine nitration, the nitrated tyrosine residues were correlated with the specific functional domains in each nitrated protein. The functional domains and motifs of each nitrated protein were then determined using the Scan prosite software and the Motif scan software, respectively (http://www.expasy.ch/tools/scanprosite/). More information can be obtained from the Swiss-Prot annotation page (http://www.uniprot.org) and Human Protein Reference Database (http://www.hprd.org) of each protein. Bioinformatic analysis revealed that tyrosine nitration occurred within the specific functional domains of the two nitrated proteins (Table 2). Further analyses of the secondary structure in the tyrosine nitration sites were performed using the NetsurfP software (http://www.cbs. dtu.dk/services/NetSurfP/).17 Caspase recruitment domain (CARD) protein 9 (CARD9) is included in the coiled-coil CARD groups that contain the CARD domain and coiled-coil domain, but not the nucleotide binding domain. This group of proteins appear to activate NF-κB through recruitment of the bipartite-CARD protein, BCL10, resulting from activation of the IKK complex.18 Similarly, CARD9 also interacts with the activation CARD domain of BCL10 (17) Petersen, B.; Petersen, T. N.; Andersen, P.; Nielsen, M.; Lundegaard, C. BMC Struct. Biol. 2009, 9, 51. (18) Willis, T. G.; Jadayel, D. M.; Du, M. Q.; Peng, H.; Perry, A. R.; Abdul-Rauf, M.; Price, H.; Karran, L.; Majekodunmi, O.; Wlodarska, I.; Pan, L.; Crook, T.; Hamoudi, R.; Isaacson, P. G.; Dyer, M. J. Cell 1999, 96, 35–45.

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Figure 4. MS/MS spectra corresponding to Huh7. (a) Hypothetical protein LOC 51306 isoform 3, H2N-PSYSDIAANPKVLK-COOH. Its total ion chromatography (TIC) value is 7.92 × 103. (b) Solute carrier family 32, member 1, H2N-FYIDVK-COOH. Its TIC value is 6.30 × 103. (c) Karyopherin β1, H2N-EHIKNPDWRYR-COOH. Its TIC value is 8.98 × 103. ]: N-terminal acetylation, ∧: lysine acetylation (mass shift of +42 Da), @: tyrosine fluorinated carbon tagging (mass shift of +289 Da).

through the CARD domain of CARD9.19 The resulting signaling complex may interact with components of the IKK complex, resulting in its activation. The nitration (Y76) located in the R helix region of the protein that occurred within the CARD domain might interfere with the interaction between BCL10 and CARD9, eventually resulting in dysregulation of the NF-κB activation pathway. Thus, the nitration of CARD9 may be functionally associated with various cell physiologies, including cell death, drug resistance, and immune response. RalA binding protein 1 (RALBP1) is a signaling protein known to be involved in clathrin-coated vesicle-mediated endocytosis of growth factor receptors that also functions as a transporter of glutathione-electrophilic conjugate (GS-E) and xenobiotics.20 Rho(19) Bertin, J.; Guo, Y.; Wang, L.; Srinivasula, S. M.; Jacobson, M. D.; Poyet, J. L.; Merriam, S.; Du, M. Q.; Dyer, M. J.; Robison, K. E.; DiStefano, P. S.; Alnemri, E. S. J. Biol. Chem. 2000, 275, 41082–41086. (20) Awasthi, S.; Singhal, S. S.; Sharma, R.; Zimniak, P.; Awasthi, Y. C. Int. J. Cancer 2003, 106, 635–646.

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GTPase-activating protein domain interacts preferentially with Rho family member CDC42. CDC42 has been shown to control the cell morphogenesis and actin dynamics.21 Activation of CDC42 through binding with RALBP1 shows resistance to chemotherapeutic agents and radiation therapy.19 Because the nitration (Y247) occurred within this domain, and this site was located in the R helix region of the protein, nitration of RALBP1 might be functionally associated with growth factor receptor-mediated signal transduction and drug resistance of cancer. In this study, we identified several tyrosine-nitrated proteins that are known to be involved in important cellular functions such as cell maintenance, apoptosis, and proliferation. Among these proteins, two share tyrosine nitration sites with functionally known domains as summarized in Table 2. These data indicate that the tyrosine nitration might participate in the intrinsic activity of (21) Tang, Y.; Olufemi, L.; Wang, M. T.; Nie, D. Front. Biosci. 2008, 13, 759– 776.

Figure 5. Classification of nitroproteomes. (a) Molecular function and (b) biological process of enriched nitroproteins. On the basis of these findings, it is predicted that nitroproteins participated in diverse biological processes. Table 2. Information Describing Proteins That Share the Tyrosine Nitration Site with the Known Functionally Active Domain protein name

Tyr nitration site

functional domain site

caspase recruitment domain protein 9 isoform 1 RalA binding protein 1

76

14-100

caspase recruitment domain

Q9H257

247

192-380

Rho-GTPase-activating protein domain

Q15311

nitrated proteins. Although nitration has been reported to compete with phosphorylation,22 the specific role of tyrosine nitration on each protein activity has yet to be elucidated. CONCLUSIONS We introduced a new nitrotyrosine enrichment method based on a series of chemical conversions and solid-phase capture/ release for the detection and characterization of protein tyrosine nitration. Using this approach, which relies on the use of fluorine-fluorine affinity purification following chemical conversions of nitro groups on tyrosine residues to highly fluorinated moieties, we were able to selectively enrich and characterize 28 nitrated proteins from the whole proteome of the human hepatoma cell line, Huh7. Thus, this method would provide new avenues for future studies investigating important molecular links between protein tyrosine nitration and its biological functions. (22) Csibi, A.; Communi, D.; Muller, N.; Bottari, S. P. PLoS One 2010, 5, e10070.

domain name

Swiss-prot no.

ACKNOWLEDGMENT This study was supported by grants from the 21C Frontier Functional Proteomics Project (FPR08A1-032), Stem Cell Research Center of the 21st Century Frontier Research Program (SC-2120), and the Converging Research Center Program (2009-0093622) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology.

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 August 5, 2010. Accepted November 19, 2010. AC102080D Analytical Chemistry, Vol. 83, No. 1, January 1, 2011

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