MASS SPECTROMETRIC ANALYSIS OF PROTEIN TYROSINE NITRATION IN AGING AND NEURODEGENERATIVE DISEASES Woon-Seok Yeo,1 Young Jun Kim,2 Mohammad Humayun Kabir,3 Jeong Won Kang,3 and Kwang Pyo Kim3* 1
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea 2 Department of Applied Biochemistry, Konkuk University, Chungju 380-701, Republic of Korea 3 Department of Applied Chemistry, Kyung Hee University, Yongin 446-701, Republic of Korea
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21429
This review highlights the significance of protein tyrosine nitration (PTN) in signal transduction pathways, the progress achieved in analytical methods, and the implication of nitration in the cellular pathophysiology of aging and age-related neurodegenerative diseases. Although mass spectrometry of nitrated peptides has become a powerful tool for the characterization of nitrated peptides, the low stoichiometry of this modification clearly necessitates the use of affinity chromatography to enrich modified peptides. Analysis of nitropeptides involves identification of endogenous, intact modification as well as chemical conversion of the nitro group to a chemically reactive amine group and further modifications that enable affinity capture and enhance detectability by altering molecular properties. In this review, we focus on the recent progress in chemical derivatization of nitropeptides for enrichment and mass analysis, and for detection and quantification using various analytical tools. PTN participates in physiological processes, such as aging and neurodegenerative diseases. Accumulation of 3-nitrotyrosine has been found to occur during the aging process; this was identified through mass spectrometry. Further, there are several studies implicating the presence of nitrated tyrosine in age-related diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. # 2014 Wiley Periodicals, Inc. Mass Spec Rev. Keywords: protein tyrosine nitration; 3-nitrotyrosine; reactive nitrogen species; aging; neurodegeneration
Contract grant sponsor: Research Centers Program through the National Research Foundation (NRF), Korean Ministry of Education; Contract grant number: 2009-0093824; Contract grant sponsor: Proteogenomic Research Program of the NRF, Korean Ministry of Science, ICT and Future Planning; Contract grant sponsor: National Project for Personalized Genomic Medicine, Korean Ministry for Health & Welfare; Contract grant number: A111218-CP02. Correspondence to: Kwang Pyo Kim, Department of Applied Chemistry, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea. E-mail: [email protected]
Mass Spectrometry Reviews # 2014 by Wiley Periodicals, Inc.
I. INTRODUCTION Most proteins that are translated from mRNA undergo chemical modifications that help them to become functional in different body cells. Post-translational modifications (PTMs) introduce heterogeneity in proteins and enable the utilization of identical proteins for different cellular functions in different cell types (Mann et al., 2002; Manning et al., 2002). PTMs play an important role in the regulation of protein function through the modification of protein structure, activity, turnover, localization, hydrophobicity, hydrogen bonding, electrostatic properties, and the nature of protein–protein complexes (Gruhler et al., 2005; Salih, 2005; Reynolds, Berry, & Binder, 2007; Schreiber et al., 2008; Thingholm, Jensen, & Larsen, 2009). Nitration at tyrosine residues in proteins is a prominent PTM event. Protein tyrosine nitration (PTN) is a physiological event responsible for various biological processes that are caused by nitric oxide (NO) (Schopfer, Baker, & Freeman, 2003; Lee et al., 2009a). PTN involves a cascade of oxidation processes in which tyrosine residues are selectively modified. PTN is mediated by reactive nitrogen species (RNS), which are generated by the reaction of NO with reactive oxygen species (ROS). PTN has been reported to be involved in the pathogenesis (Greenacre & Ischiropoulos, 2001; Kanski, Hong, & Schoneich, 2005b) of numerous inflammatory responses (Kanski & Schoneich, 2005), cytoskeletal dysfunction (Nonnis et al., 2008), platelet activation (Sabetkar et al., 2008), agerelated diseases such as age-related macular degeneration (AMD) (Murdaugh et al., 2010), neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) (Beal, 2002; Turko & Murad, 2002; Fahn, 2003; Ischiropoulos & Beckman, 2003; Schopfer, Baker, & Freeman, 2003; Gow et al., 2004; Sacksteder et al., 2006; Sawada et al., 2007; Lee et al., 2009a), cardiovascular disease, stroke, decreased immune responses (Ames, 1995; Beal, 1995), and even cancer (Kim et al., 2011). It has been reported that the redox regulation of normal metabolism is harmonized by the increased abundance of nitrated proteins (Sacksteder et al., 2006). Protein nitration contributes to cellular signaling mechanisms (Di Stasi et al., 1999; Squier & Bigelow, 2000) by its properties of specificity, reversibility, and controlled rates of formation and modification of target proteins and cell functions (Gow et al., 1996; Kamisaki et al., 1998). PTN also has certain major specific effects, such as affecting protein structure and function, modulating phosphorylation cascades, and occasionally inducing an immune response. The
&
YEO ET AL.
nitration of tyrosine residues to 3-nitrotyrosine (3-NT) precludes the ability of tyrosine residues to undergo cyclic introversions between their phosphorylated and un-phosphorylated forms (Hunter, 1995). Though our focus is on the recent progress in chemical derivatization of nitropeptides for enrichment and mass spectrometric analysis, and for detection and quantification, various analytical tools were employed to analyze PTN without chemical derivatization. Studies were performed to identify endogenous nitroproteins from post mortem human pituitary and mouse brain where two-dimensional gel electrophoresis (2DGE)-based Western blot analysis was used to separate endogenous nitroproteins from the pituitary tissue sample. Nitroproteins separated by 2DGE were confirmed by Western blotting with anti-nitrotyrosine antibody. Excised nitroprotein spots were subjected to trypsin digestion followed by the amino acid sequence analysis with LC-MS/MS to identify nitroproteins and nitrotyrosine sites (Zhan & Desiderio, 2004; Sacksteder et al., 2006). Another study was done by the same group to characterize endogenous nitroproteins from human pituitary adenoma. In this case a nitrotyrosine affinity column (NTAC) was used to exclusively enrich and isolate endogenous nitroproteins from a tissue homogenate. NTAC is an effective method to isolate and enrich nitroproteins from a complicated proteome sample to improve tandem mass spectrometry (MS/MS) identification of very low-abundance nitroproteins. In this method, antinitrotyrosine antibodies were cross-linked to protein G beads and then incubated with the pituitary adenoma protein sample. Nitroproteins were bound to the cross-linked anti-nitrotyrosine antibodies which, later, were eluted to provide an enriched nitroprotein sample. The enriched endogenous nitroprotein complexes were subjected to trypsin digestion, desalination, and MS/MS analysis (Zhan & Desiderio, 2006). The third method involves identifying human chronic obstructive pulmonary disease (COPD)-related bronchoalveolar lavage fluid (BALF) nitroproteins, nitroproteome in neuron-like PC12 cells or in mouse brains. Nitroproteins were immunoprecipitated and the protein samples were then digested with trypsin and finally the tryptic peptides were analyzed with matrix-assisted laser desorption/ionization (MALDI)-MS/MS (Tedeschi et al., 2005; Nonnis et al., 2008; Zhan & Desiderio, 2011). In this article, we attempt to describe the PTN information such as mechanism, signal transduction, enrichment and detection, and association with age-related diseases. Figure 1 shows the scheme to composition of this article.
II. REACTIVE NITROGEN SPECIES Radical and non-radical RNS include NO, nitrogen dioxide (NO2), nitrous acid (HNO2), nitrosyl cation (NOþ), nitrosyl anion (NO), dinitrogen tetroxide (N2O4), dinitrogen trioxide (N2O3), peroxynitrite (PN, ONOO), peroxynitrous acid (ONOOH), alkyl peroxynitrites (ROONO), nitronium cation (NO2þ), and nitrylchloride (NO2Cl). Several ROS are also present in the biological system. Protein nitration is the result of several combinatorial reactions of RNS and ROS (Turko & Murad, 2002). Nitric oxide (NO) is a short-lived physiological messenger that is highly diffusible and lipophilic (Masri, 2010) in nature. NO regulates several significant physiological functions, including vasodilation, respiration, cell migration, immune responses, and apoptosis (Muntane & la Mata, 2010). NO is produced within cells by the actions of nitric oxide synthases (NOS) 2
FIGURE 1. This article was composed to six parts such as reactive nitrogen species, signal transduction, mass spectrometric analysis, and relation with age-related diseases.
(Drew & Leeuwenburgh, 2002). There are three distinct isoforms of NOS: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3) (Ignarro et al., 1987; Nathan, 1992; Muntane & la Mata, 2010). All these isoforms exhibit similar structures and catalytic modes but differ in their mechanism of activity. Several cofactors such as NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) are involved in the catalysis of NO production by NOS (Ignarro et al., 2001; Muntane & la Mata, 2010). Following stimulation by immunological or inflammatory reactions, a cytokine-induced NOS isoform is expressed in many cells, including macrophages and hepatocytes, and produces large amounts of NO (Moncada, Palmer, & Higgs, 1991). All NOSs require similar amounts of L-arginine, NADPH, and BH4 for their activity. These NOS isoforms are regulated differentially at the transcriptional, translational, and post-translational levels. The activities of NOS-1 and NOS-3 are highly dependent upon the intracellular Ca2þ concentration, whereas NOS-2 forms an active complex with calmodulin (CaM), and is already maximally activated by Ca2þ/CaM even at basal levels of intracellular Ca2þ (Alderton, Cooper, & Knowles, 2001). Several inhibitory and activator phosphorylated sites in NOS-1 and NOS-3 tightly regulate their NO production (Church & Fulton, 2006). Intracellular localization indicates the relevance of NOS activity; NOSs are specifically targeted to subcellular compartments (plasma membrane, golgi, cytosol, nucleus, and mitochondria) and this trafficking is crucial for NO production and specific PTMs of target proteins. The biological lifetime of NO is approximately 5 sec; it can carry out its function as an intracellular as well as an extracellular messenger by diffusing across distances equivalent to several cell diameters within this short time (Sohal & Orr, 1992; Ames, Shigenaga, & Hagen, 1993; Lancaster, 1994). Mass Spectrometry Reviews DOI 10.1002/mas
MASS SPECTROMETRY OF PROTEIN TYROSINE NITRATION
&
Several chemical modifications occur in protein tyrosine residues, including nitration, chlorination, and bromination. Tyrosine residues in protein can be chlorinated to form 3chlorotyrosine by HOCl, brominated to form 3-bromotyrosine by HOBr (Aldridge et al., 2002), or nitrated by NO and its metabolites to form 3-NT (defined as protein nitration) (Beckman et al., 1992; Haddad et al., 1994). PTN is a hallmark of oxidative stress and is modulated by RNS (Ignarro, 1990; Haddad et al., 1994; MacMillan-Crow et al., 1996; Masri, 2010) such as PN and nitrogen dioxide (NO2). These are secondary products of NO metabolism generated in the presence of oxidants, including superoxide radicals (O2•), hydrogen peroxide (H2O2), and transition metal centers (Radi, 2004). Nitric oxide, being a relatively un-reactive radical, is able to form other reactive intermediates that can trigger nitrosative damage on biomolecules, which in turn may lead to age-related diseases due to the structural alteration of proteins, inhibition of enzymatic activity, and interference with regulatory functions. Simplified possible pathways to PTN are illustrated in Figure 2.
III. SIGNAL TRANSDUCTION BY PROTEIN TYROSINE NITRATION: COMPETITION OR COOPERATION WITH TYROSINE PHOSPHORYLATION-DEPENDENT SIGNALING PATHWAY Introduction of a nitro group (NO2) into a chemical compound through a chemical process is generally known as nitration. In the case of proteins, several amino acids, such as tyrosine, tryptophan, cysteine, and methionine, are preferentially nitrated. However, most studies concern tyrosine nitration, which consists of the addition of a nitro group to one of the two equivalent ortho-carbons of the aromatic ring of tyrosine residues, changing tyrosine into a negatively charged hydrophilic nitrotyrosine moiety and causing a marked shift in the local pKa of the hydroxyl group from 10.07 in tyrosine to 7.50 in nitrotyrosine (Turko & Murad, 2002; Corpas et al., 2009). Tyrosine nitration is a very selective process. Only 1–2% out of the total 3–4 mol% of tyrosines (Tyr) may become preferentially nitrated, depending on the structure, nitration mechanism, and environment of the protein (Bartesaghi et al., 2007). Another published data reports that 0–1 nitrated residues per 106 tyrosine residues are observed in normal rat plasma (Shigenaga et al., 1997). Tyrosine nitration is capable of causing gain-of-function as well as no effect on function; however, inhibition of function is the most common consequence of PTN (Radi, 2004). Nitration of a tyrosine residue may either prevent further phosphorylation or stimulate phosphorylation (Rayala et al., 2007; Shi et al., 2007). Several experiments have been conducted to elucidate if nitration collaborates or competes with phosphorylation. Kong et al. concluded that the PN-mediated nitration of a single tyrosine residue prevents tyrosine phosphorylation in purified cdc2, a cell cycle kinase (Kong et al., 1996). Another research team tested bovine pulmonary arteries exposed to PN and observed decreased levels of tyrosine-phosphorylated proteins and increased levels of nitrotyrosine-containing proteins (Gow et al., 1996). These observations indicate that the importance of tyrosine nitration in cell signaling lies essentially in the inhibition of tyrosine residues from undergoing cyclic conversions between phosphorylated and non-phosphorylated states. On the other hand, several other experiments have shown that
Mass Spectrometry Reviews DOI 10.1002/mas
FIGURE 2. Pathways of protein tyrosine nitration. In aqueous environments, peroxynitrite (ONOO) reacts with CO2 to generate nitrosoperoxocarboxylate (ONOOCO2), which readily decomposes to carbonate radicals (CO3•) and nitric dioxide (NO2) by hemolytic cleavage and contributes to the process of tyrosine nitration[1]. Unstable peroxynitrous acid (ONOOH) is generated from the protonation of ONOO at suitable pH. Hemolytic cleavage of ONOOH produces hydroxyl radicals (•OH) and NO2 that also can participate in direct reactions of tyrosine nitration[2]. On the other hand, NO can be oxidized to the nitrite ion (NO2), which is converted to NO2. Peroxidases, such as myeloperoxidase, metabolize NO2 and hydrogen peroxide (H2O2) to generate OH and NO2 and subsequently lead to tyrosine nitration[3] (Lee et al., 2009a).
tyrosine nitration can also promote tyrosine phosphorylation (Li et al., 1998; Di Stasi et al., 1999). These observations indicate that protein tyrosine phosphorylation and PTN are mutually exclusive events. However, Mallozzi et al. showed that a nitrotyrosine imitates the phosphotyrosine binding site in the SH2 domain of the src family tyrosine kinase lyn (Mallozzi, Di Stasi, & Minetti, 2001). Further, in vitro induction of both phosphorylation and nitration is reported by PN in recombinant non-active GST-MEK1 (Zhang et al., 2000). Phosphotyrosine-dependent signaling may be either positively or negatively regulated via the nitration of tyrosine residues in proteins (Turko & Murad, 2002). Angiotensin II stimulation causes tyrosine nitration in extracellular signal-related kinase (ERK) 1/2 in rat vascular smooth muscle cells (VSMCs). Immunoprecipitation of phospho-ERK1/2 with an anti-nitrotyrosine antibody suggests that the sites are distinct. Angiotensin II, via the activation of the AT1 receptor, induces the production of ROS and RNS with the consequent formation of PN. This, in turn, nitrates tyrosine residues in ERK1/2 and hence facilitates the activation of phosphorylation. In parallel with the induction of a redoxsensitive pathway that contributes to the nitration and activation of ERK, angiotensin II also activates the Raf–MEK–ERK pathway that is required for ERK phosphorylation and activation (Pinzar et al., 2005). 3
&
YEO ET AL.
IV. SELECTIVITY OF PROTEIN TYROSINE NITRATION Nitration on tyrosine residues is a functionally important process; hence only selective tyrosine residues are nitrated within a protein, and thus all proteins may not be necessarily nitrated in vivo. Souza et al. investigated factors that may determine the biological selectivity of PTN with an in vitro model consisting of three proteins of similar sizes but different three-dimensional structures and tyrosine contents. The factors they considered were the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge. This study revealed several sequence and structural observations regarding nitration of tyrosine residues in exogenous proteins, although the factors of nitration in vitro are not always the same as in vivo. First, all tyrosine residues are not available for nitration. The tyrosine residues that are not exposed to solvent phase are not available for nitration. Tyrosine residues do not pack into the interior of proteins like phenylalanine, tryptophan, and non-polar side-chain amino acids. Second, no apparent sequence homology is available to be recognized by different nitrating agents. Sequences spanning 5 to þ5 amino acid residues relative to the nitrated tyrosines of different proteins after nitration by PN did not exhibit any specific sequence pattern. Yet, within the same span of residues, the presence of one or more acidic residues and the relative paucity of cysteine, methionine, and basic amino acids were noted. Third, almost all of the tyrosine residues that can be nitrated are found in loop structures; therefore, the secondary structure of the protein and the local environment of the tyrosine residue may play significant roles in determining the site of nitration. Fourth, the selection of different nitrating agents varies with different protein targets. Fifth, the preferred protein target for nitration is not dependent on the number of tyrosine residues in a protein or on the concentration of the protein in the solution (Souza et al., 1999). Elfering et al. constructed a consensus nitration target sequence as follows: H-X-[DE]-H-X(2,3)-H(2)-X(2,4)-Y, in which H represents a hydrophobic residue (such as L, M, V, I, P, A, F, or W), X is any amino acid, and Y is the target tyrosine, and reported that tyrosine residues in hydrophobic pockets are preferred targets for nitration. Additionally, this study also noted enhancement of tyrosine nitration in the proximity of acidic residues (i.e., the presence of D or E) (Elfering et al., 2004). Zhang et al. also showed that the site selectivity of exogenous protein nitration depends on the nitrating agent, reaction conditions, and molecular structure (primary, secondary, and tertiary) of the protein (Zhang, Yang, & Poschl, 2011).
V. MASS SPECTROMETRIC ANALYSIS OF NITRATED PEPTIDES A. Factors Contribute Misidentification of Tyrosine Nitration and Actions Required for Correction Standard data-dependent acquisition on a quadrupole or linear ion trap usually employs low mass accuracy-based methods. The subsequent database search applies a 2 3-Da precursor ion mass tolerance. As a result, false positive identification of peptide modifications may occur when processing a large quantity of data through a database search algorithm. Additionally, factors 4
such as carbamoylation of the N terminus or basic residues in the peptide sequence, deamidation, 13C-based data-dependent isotope selection, random noise spikes, modification-specific fragmentation, or the selection of co-eluting isobaric peptide ions may confound the analysis of tandem mass spectrometry (MS/ MS) spectra. In addition, the definitive assignment of tyrosine nitration may be difficult due to “b-” or “y-” ion coverage, which would afford sequence information about the N-terminal portion of the peptide if carbamoylation occurs at this particular region. Therefore, the collection of multiple MS/MS spectra for a specific peptide ion during data-dependent acquisition in mass spectrometry will improve identification accuracy. Further, the presence of random noise or isobaric co-eluting peptides must be confirmed. The b- and y-ion series should be checked carefully to determine whether they are derived from a “real” peptide of the analyzed sample. “Background ions” may appear in the MS spectrum or selected as precursor ion for MS/MS analysis (Zhan & Desiderio, 2009). For modification-specific fragmentation, retention of the nitration modification on the tyrosine residue during collision-induced dissociation (CID) fragmentation, with no further fragmentation of the actual NO2 group of the nitrated peptide, should be monitored. Consequently, manual evaluation of MS/MS spectra to identify peptide nitration or other modification is considered an important step. It is noted that manual analysis of MS/MS spectra should have at least 20–25% relative intensity. Additionally, with respect to failing to resolve peptide modifications, nitrated peptides can be synthesized and analyzed by MS/MS using similar experimental conditions. Finally, short peptide sequences of
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea 2 Department of Applied Biochemistry, Konkuk University, Chungju 380-701, Republic of Korea 3 Department of Applied Chemistry, Kyung Hee University, Yongin 446-701, Republic of Korea
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mas.21429
This review highlights the significance of protein tyrosine nitration (PTN) in signal transduction pathways, the progress achieved in analytical methods, and the implication of nitration in the cellular pathophysiology of aging and age-related neurodegenerative diseases. Although mass spectrometry of nitrated peptides has become a powerful tool for the characterization of nitrated peptides, the low stoichiometry of this modification clearly necessitates the use of affinity chromatography to enrich modified peptides. Analysis of nitropeptides involves identification of endogenous, intact modification as well as chemical conversion of the nitro group to a chemically reactive amine group and further modifications that enable affinity capture and enhance detectability by altering molecular properties. In this review, we focus on the recent progress in chemical derivatization of nitropeptides for enrichment and mass analysis, and for detection and quantification using various analytical tools. PTN participates in physiological processes, such as aging and neurodegenerative diseases. Accumulation of 3-nitrotyrosine has been found to occur during the aging process; this was identified through mass spectrometry. Further, there are several studies implicating the presence of nitrated tyrosine in age-related diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. # 2014 Wiley Periodicals, Inc. Mass Spec Rev. Keywords: protein tyrosine nitration; 3-nitrotyrosine; reactive nitrogen species; aging; neurodegeneration
Contract grant sponsor: Research Centers Program through the National Research Foundation (NRF), Korean Ministry of Education; Contract grant number: 2009-0093824; Contract grant sponsor: Proteogenomic Research Program of the NRF, Korean Ministry of Science, ICT and Future Planning; Contract grant sponsor: National Project for Personalized Genomic Medicine, Korean Ministry for Health & Welfare; Contract grant number: A111218-CP02. Correspondence to: Kwang Pyo Kim, Department of Applied Chemistry, Kyung Hee University, 1732 Deogyeong-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-701, Republic of Korea. E-mail: [email protected]
Mass Spectrometry Reviews # 2014 by Wiley Periodicals, Inc.
I. INTRODUCTION Most proteins that are translated from mRNA undergo chemical modifications that help them to become functional in different body cells. Post-translational modifications (PTMs) introduce heterogeneity in proteins and enable the utilization of identical proteins for different cellular functions in different cell types (Mann et al., 2002; Manning et al., 2002). PTMs play an important role in the regulation of protein function through the modification of protein structure, activity, turnover, localization, hydrophobicity, hydrogen bonding, electrostatic properties, and the nature of protein–protein complexes (Gruhler et al., 2005; Salih, 2005; Reynolds, Berry, & Binder, 2007; Schreiber et al., 2008; Thingholm, Jensen, & Larsen, 2009). Nitration at tyrosine residues in proteins is a prominent PTM event. Protein tyrosine nitration (PTN) is a physiological event responsible for various biological processes that are caused by nitric oxide (NO) (Schopfer, Baker, & Freeman, 2003; Lee et al., 2009a). PTN involves a cascade of oxidation processes in which tyrosine residues are selectively modified. PTN is mediated by reactive nitrogen species (RNS), which are generated by the reaction of NO with reactive oxygen species (ROS). PTN has been reported to be involved in the pathogenesis (Greenacre & Ischiropoulos, 2001; Kanski, Hong, & Schoneich, 2005b) of numerous inflammatory responses (Kanski & Schoneich, 2005), cytoskeletal dysfunction (Nonnis et al., 2008), platelet activation (Sabetkar et al., 2008), agerelated diseases such as age-related macular degeneration (AMD) (Murdaugh et al., 2010), neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) (Beal, 2002; Turko & Murad, 2002; Fahn, 2003; Ischiropoulos & Beckman, 2003; Schopfer, Baker, & Freeman, 2003; Gow et al., 2004; Sacksteder et al., 2006; Sawada et al., 2007; Lee et al., 2009a), cardiovascular disease, stroke, decreased immune responses (Ames, 1995; Beal, 1995), and even cancer (Kim et al., 2011). It has been reported that the redox regulation of normal metabolism is harmonized by the increased abundance of nitrated proteins (Sacksteder et al., 2006). Protein nitration contributes to cellular signaling mechanisms (Di Stasi et al., 1999; Squier & Bigelow, 2000) by its properties of specificity, reversibility, and controlled rates of formation and modification of target proteins and cell functions (Gow et al., 1996; Kamisaki et al., 1998). PTN also has certain major specific effects, such as affecting protein structure and function, modulating phosphorylation cascades, and occasionally inducing an immune response. The
&
YEO ET AL.
nitration of tyrosine residues to 3-nitrotyrosine (3-NT) precludes the ability of tyrosine residues to undergo cyclic introversions between their phosphorylated and un-phosphorylated forms (Hunter, 1995). Though our focus is on the recent progress in chemical derivatization of nitropeptides for enrichment and mass spectrometric analysis, and for detection and quantification, various analytical tools were employed to analyze PTN without chemical derivatization. Studies were performed to identify endogenous nitroproteins from post mortem human pituitary and mouse brain where two-dimensional gel electrophoresis (2DGE)-based Western blot analysis was used to separate endogenous nitroproteins from the pituitary tissue sample. Nitroproteins separated by 2DGE were confirmed by Western blotting with anti-nitrotyrosine antibody. Excised nitroprotein spots were subjected to trypsin digestion followed by the amino acid sequence analysis with LC-MS/MS to identify nitroproteins and nitrotyrosine sites (Zhan & Desiderio, 2004; Sacksteder et al., 2006). Another study was done by the same group to characterize endogenous nitroproteins from human pituitary adenoma. In this case a nitrotyrosine affinity column (NTAC) was used to exclusively enrich and isolate endogenous nitroproteins from a tissue homogenate. NTAC is an effective method to isolate and enrich nitroproteins from a complicated proteome sample to improve tandem mass spectrometry (MS/MS) identification of very low-abundance nitroproteins. In this method, antinitrotyrosine antibodies were cross-linked to protein G beads and then incubated with the pituitary adenoma protein sample. Nitroproteins were bound to the cross-linked anti-nitrotyrosine antibodies which, later, were eluted to provide an enriched nitroprotein sample. The enriched endogenous nitroprotein complexes were subjected to trypsin digestion, desalination, and MS/MS analysis (Zhan & Desiderio, 2006). The third method involves identifying human chronic obstructive pulmonary disease (COPD)-related bronchoalveolar lavage fluid (BALF) nitroproteins, nitroproteome in neuron-like PC12 cells or in mouse brains. Nitroproteins were immunoprecipitated and the protein samples were then digested with trypsin and finally the tryptic peptides were analyzed with matrix-assisted laser desorption/ionization (MALDI)-MS/MS (Tedeschi et al., 2005; Nonnis et al., 2008; Zhan & Desiderio, 2011). In this article, we attempt to describe the PTN information such as mechanism, signal transduction, enrichment and detection, and association with age-related diseases. Figure 1 shows the scheme to composition of this article.
II. REACTIVE NITROGEN SPECIES Radical and non-radical RNS include NO, nitrogen dioxide (NO2), nitrous acid (HNO2), nitrosyl cation (NOþ), nitrosyl anion (NO), dinitrogen tetroxide (N2O4), dinitrogen trioxide (N2O3), peroxynitrite (PN, ONOO), peroxynitrous acid (ONOOH), alkyl peroxynitrites (ROONO), nitronium cation (NO2þ), and nitrylchloride (NO2Cl). Several ROS are also present in the biological system. Protein nitration is the result of several combinatorial reactions of RNS and ROS (Turko & Murad, 2002). Nitric oxide (NO) is a short-lived physiological messenger that is highly diffusible and lipophilic (Masri, 2010) in nature. NO regulates several significant physiological functions, including vasodilation, respiration, cell migration, immune responses, and apoptosis (Muntane & la Mata, 2010). NO is produced within cells by the actions of nitric oxide synthases (NOS) 2
FIGURE 1. This article was composed to six parts such as reactive nitrogen species, signal transduction, mass spectrometric analysis, and relation with age-related diseases.
(Drew & Leeuwenburgh, 2002). There are three distinct isoforms of NOS: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3) (Ignarro et al., 1987; Nathan, 1992; Muntane & la Mata, 2010). All these isoforms exhibit similar structures and catalytic modes but differ in their mechanism of activity. Several cofactors such as NADPH, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and tetrahydrobiopterin (BH4) are involved in the catalysis of NO production by NOS (Ignarro et al., 2001; Muntane & la Mata, 2010). Following stimulation by immunological or inflammatory reactions, a cytokine-induced NOS isoform is expressed in many cells, including macrophages and hepatocytes, and produces large amounts of NO (Moncada, Palmer, & Higgs, 1991). All NOSs require similar amounts of L-arginine, NADPH, and BH4 for their activity. These NOS isoforms are regulated differentially at the transcriptional, translational, and post-translational levels. The activities of NOS-1 and NOS-3 are highly dependent upon the intracellular Ca2þ concentration, whereas NOS-2 forms an active complex with calmodulin (CaM), and is already maximally activated by Ca2þ/CaM even at basal levels of intracellular Ca2þ (Alderton, Cooper, & Knowles, 2001). Several inhibitory and activator phosphorylated sites in NOS-1 and NOS-3 tightly regulate their NO production (Church & Fulton, 2006). Intracellular localization indicates the relevance of NOS activity; NOSs are specifically targeted to subcellular compartments (plasma membrane, golgi, cytosol, nucleus, and mitochondria) and this trafficking is crucial for NO production and specific PTMs of target proteins. The biological lifetime of NO is approximately 5 sec; it can carry out its function as an intracellular as well as an extracellular messenger by diffusing across distances equivalent to several cell diameters within this short time (Sohal & Orr, 1992; Ames, Shigenaga, & Hagen, 1993; Lancaster, 1994). Mass Spectrometry Reviews DOI 10.1002/mas
MASS SPECTROMETRY OF PROTEIN TYROSINE NITRATION
&
Several chemical modifications occur in protein tyrosine residues, including nitration, chlorination, and bromination. Tyrosine residues in protein can be chlorinated to form 3chlorotyrosine by HOCl, brominated to form 3-bromotyrosine by HOBr (Aldridge et al., 2002), or nitrated by NO and its metabolites to form 3-NT (defined as protein nitration) (Beckman et al., 1992; Haddad et al., 1994). PTN is a hallmark of oxidative stress and is modulated by RNS (Ignarro, 1990; Haddad et al., 1994; MacMillan-Crow et al., 1996; Masri, 2010) such as PN and nitrogen dioxide (NO2). These are secondary products of NO metabolism generated in the presence of oxidants, including superoxide radicals (O2•), hydrogen peroxide (H2O2), and transition metal centers (Radi, 2004). Nitric oxide, being a relatively un-reactive radical, is able to form other reactive intermediates that can trigger nitrosative damage on biomolecules, which in turn may lead to age-related diseases due to the structural alteration of proteins, inhibition of enzymatic activity, and interference with regulatory functions. Simplified possible pathways to PTN are illustrated in Figure 2.
III. SIGNAL TRANSDUCTION BY PROTEIN TYROSINE NITRATION: COMPETITION OR COOPERATION WITH TYROSINE PHOSPHORYLATION-DEPENDENT SIGNALING PATHWAY Introduction of a nitro group (NO2) into a chemical compound through a chemical process is generally known as nitration. In the case of proteins, several amino acids, such as tyrosine, tryptophan, cysteine, and methionine, are preferentially nitrated. However, most studies concern tyrosine nitration, which consists of the addition of a nitro group to one of the two equivalent ortho-carbons of the aromatic ring of tyrosine residues, changing tyrosine into a negatively charged hydrophilic nitrotyrosine moiety and causing a marked shift in the local pKa of the hydroxyl group from 10.07 in tyrosine to 7.50 in nitrotyrosine (Turko & Murad, 2002; Corpas et al., 2009). Tyrosine nitration is a very selective process. Only 1–2% out of the total 3–4 mol% of tyrosines (Tyr) may become preferentially nitrated, depending on the structure, nitration mechanism, and environment of the protein (Bartesaghi et al., 2007). Another published data reports that 0–1 nitrated residues per 106 tyrosine residues are observed in normal rat plasma (Shigenaga et al., 1997). Tyrosine nitration is capable of causing gain-of-function as well as no effect on function; however, inhibition of function is the most common consequence of PTN (Radi, 2004). Nitration of a tyrosine residue may either prevent further phosphorylation or stimulate phosphorylation (Rayala et al., 2007; Shi et al., 2007). Several experiments have been conducted to elucidate if nitration collaborates or competes with phosphorylation. Kong et al. concluded that the PN-mediated nitration of a single tyrosine residue prevents tyrosine phosphorylation in purified cdc2, a cell cycle kinase (Kong et al., 1996). Another research team tested bovine pulmonary arteries exposed to PN and observed decreased levels of tyrosine-phosphorylated proteins and increased levels of nitrotyrosine-containing proteins (Gow et al., 1996). These observations indicate that the importance of tyrosine nitration in cell signaling lies essentially in the inhibition of tyrosine residues from undergoing cyclic conversions between phosphorylated and non-phosphorylated states. On the other hand, several other experiments have shown that
Mass Spectrometry Reviews DOI 10.1002/mas
FIGURE 2. Pathways of protein tyrosine nitration. In aqueous environments, peroxynitrite (ONOO) reacts with CO2 to generate nitrosoperoxocarboxylate (ONOOCO2), which readily decomposes to carbonate radicals (CO3•) and nitric dioxide (NO2) by hemolytic cleavage and contributes to the process of tyrosine nitration[1]. Unstable peroxynitrous acid (ONOOH) is generated from the protonation of ONOO at suitable pH. Hemolytic cleavage of ONOOH produces hydroxyl radicals (•OH) and NO2 that also can participate in direct reactions of tyrosine nitration[2]. On the other hand, NO can be oxidized to the nitrite ion (NO2), which is converted to NO2. Peroxidases, such as myeloperoxidase, metabolize NO2 and hydrogen peroxide (H2O2) to generate OH and NO2 and subsequently lead to tyrosine nitration[3] (Lee et al., 2009a).
tyrosine nitration can also promote tyrosine phosphorylation (Li et al., 1998; Di Stasi et al., 1999). These observations indicate that protein tyrosine phosphorylation and PTN are mutually exclusive events. However, Mallozzi et al. showed that a nitrotyrosine imitates the phosphotyrosine binding site in the SH2 domain of the src family tyrosine kinase lyn (Mallozzi, Di Stasi, & Minetti, 2001). Further, in vitro induction of both phosphorylation and nitration is reported by PN in recombinant non-active GST-MEK1 (Zhang et al., 2000). Phosphotyrosine-dependent signaling may be either positively or negatively regulated via the nitration of tyrosine residues in proteins (Turko & Murad, 2002). Angiotensin II stimulation causes tyrosine nitration in extracellular signal-related kinase (ERK) 1/2 in rat vascular smooth muscle cells (VSMCs). Immunoprecipitation of phospho-ERK1/2 with an anti-nitrotyrosine antibody suggests that the sites are distinct. Angiotensin II, via the activation of the AT1 receptor, induces the production of ROS and RNS with the consequent formation of PN. This, in turn, nitrates tyrosine residues in ERK1/2 and hence facilitates the activation of phosphorylation. In parallel with the induction of a redoxsensitive pathway that contributes to the nitration and activation of ERK, angiotensin II also activates the Raf–MEK–ERK pathway that is required for ERK phosphorylation and activation (Pinzar et al., 2005). 3
&
YEO ET AL.
IV. SELECTIVITY OF PROTEIN TYROSINE NITRATION Nitration on tyrosine residues is a functionally important process; hence only selective tyrosine residues are nitrated within a protein, and thus all proteins may not be necessarily nitrated in vivo. Souza et al. investigated factors that may determine the biological selectivity of PTN with an in vitro model consisting of three proteins of similar sizes but different three-dimensional structures and tyrosine contents. The factors they considered were the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge. This study revealed several sequence and structural observations regarding nitration of tyrosine residues in exogenous proteins, although the factors of nitration in vitro are not always the same as in vivo. First, all tyrosine residues are not available for nitration. The tyrosine residues that are not exposed to solvent phase are not available for nitration. Tyrosine residues do not pack into the interior of proteins like phenylalanine, tryptophan, and non-polar side-chain amino acids. Second, no apparent sequence homology is available to be recognized by different nitrating agents. Sequences spanning 5 to þ5 amino acid residues relative to the nitrated tyrosines of different proteins after nitration by PN did not exhibit any specific sequence pattern. Yet, within the same span of residues, the presence of one or more acidic residues and the relative paucity of cysteine, methionine, and basic amino acids were noted. Third, almost all of the tyrosine residues that can be nitrated are found in loop structures; therefore, the secondary structure of the protein and the local environment of the tyrosine residue may play significant roles in determining the site of nitration. Fourth, the selection of different nitrating agents varies with different protein targets. Fifth, the preferred protein target for nitration is not dependent on the number of tyrosine residues in a protein or on the concentration of the protein in the solution (Souza et al., 1999). Elfering et al. constructed a consensus nitration target sequence as follows: H-X-[DE]-H-X(2,3)-H(2)-X(2,4)-Y, in which H represents a hydrophobic residue (such as L, M, V, I, P, A, F, or W), X is any amino acid, and Y is the target tyrosine, and reported that tyrosine residues in hydrophobic pockets are preferred targets for nitration. Additionally, this study also noted enhancement of tyrosine nitration in the proximity of acidic residues (i.e., the presence of D or E) (Elfering et al., 2004). Zhang et al. also showed that the site selectivity of exogenous protein nitration depends on the nitrating agent, reaction conditions, and molecular structure (primary, secondary, and tertiary) of the protein (Zhang, Yang, & Poschl, 2011).
V. MASS SPECTROMETRIC ANALYSIS OF NITRATED PEPTIDES A. Factors Contribute Misidentification of Tyrosine Nitration and Actions Required for Correction Standard data-dependent acquisition on a quadrupole or linear ion trap usually employs low mass accuracy-based methods. The subsequent database search applies a 2 3-Da precursor ion mass tolerance. As a result, false positive identification of peptide modifications may occur when processing a large quantity of data through a database search algorithm. Additionally, factors 4
such as carbamoylation of the N terminus or basic residues in the peptide sequence, deamidation, 13C-based data-dependent isotope selection, random noise spikes, modification-specific fragmentation, or the selection of co-eluting isobaric peptide ions may confound the analysis of tandem mass spectrometry (MS/ MS) spectra. In addition, the definitive assignment of tyrosine nitration may be difficult due to “b-” or “y-” ion coverage, which would afford sequence information about the N-terminal portion of the peptide if carbamoylation occurs at this particular region. Therefore, the collection of multiple MS/MS spectra for a specific peptide ion during data-dependent acquisition in mass spectrometry will improve identification accuracy. Further, the presence of random noise or isobaric co-eluting peptides must be confirmed. The b- and y-ion series should be checked carefully to determine whether they are derived from a “real” peptide of the analyzed sample. “Background ions” may appear in the MS spectrum or selected as precursor ion for MS/MS analysis (Zhan & Desiderio, 2009). For modification-specific fragmentation, retention of the nitration modification on the tyrosine residue during collision-induced dissociation (CID) fragmentation, with no further fragmentation of the actual NO2 group of the nitrated peptide, should be monitored. Consequently, manual evaluation of MS/MS spectra to identify peptide nitration or other modification is considered an important step. It is noted that manual analysis of MS/MS spectra should have at least 20–25% relative intensity. Additionally, with respect to failing to resolve peptide modifications, nitrated peptides can be synthesized and analyzed by MS/MS using similar experimental conditions. Finally, short peptide sequences of
