Journal of Experimental Botany

Journal of Experimental Botany 2008 59(14):3781-3801; doi:10.1093/jxb/ern252

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REVIEW-ARTICLE

Redox proteomics: basic principles and future perspectives for the detection of protein oxidation in plants

Sara Rinalducci, Leonardo Murgiano and Lello Zolla^*

Department of Environmental Sciences, University of Tuscia, Largo dell'Università snc, I-01100, Viterbo, Italy

^* To whom correspondence should be addressed: E-mail: zolla{at}unitus.it

Received 7 July 2008; Revised 12 September 2008 Accepted 16 September 2008

	Abstract

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
The production and scavenging of chemically reactive species, such as ROS/RNS, are central to a broad range of biotic and abiotic stress and physiological responses in plants. Among the techniques developed for the identification of oxidative stress-induced modifications on proteins, the so-called ‘redox proteome’, proteomics appears to be the best-suited approach. Oxidative or nitrosative stress leaves different footprints in the cell in the form of different oxidatively modified components and, using the redox proteome, it will be possible to decipher the potential roles played by ROS/RNS-induced modifications in stressed cells. The purpose of this review is to present an overview of the latest research endeavours in the field of plant redox proteomics to identify the role of post-translational modifications of proteins in developmental cell stress. All the strategies set up to analyse the different oxidized/nitrosated amino acids, as well as the different reactivities of ROS and RNS for different amino acids are revised and discussed. A growing body of evidence indicates that ROS/RNS-induced protein modifications may be of physiological significance, and that in some cellular stresses they may act causatively and not arise as a secondary consequence of cell damage. Thus, although previously the oxidative modification of proteins was thought to represent a detrimental process in which the modified proteins were irreversibly inactivated, it is now clear that, in plants, oxidatively/nitrosatively modified proteins can be specific and reversible, playing a key role in normal cell physiology. In this sense, redox proteomics will have a central role in the definition of redox molecular mechanisms associated with cellular stresses.

Key words: Oxidative damage, oxidized/nitrosated amino acids, plant redox proteomics, protein oxidation, reactive oxygen species, reactive nitrogen species, redox regulation, stress

	Introduction

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
Oxidative stress can be defined as the toxic effect of chemically reactive species derived from (i) oxygen modification, also known as Reactive Oxygen Species (ROS), such as hydroxyl, peroxide, and superoxide radicals, or (ii) mixed nitrogen–oxygen species (RNS), such as nitric oxide (NO^•) and peroxynitrite (ONOO^–).

The different ROS have very different properties. H₂O₂ is relatively stable and its concentration in plant tissues is in the micromolar to low millimolar range, probably depending on the compartment. The other ROS have very short half-lives and are probably present at very low concentrations. The hydroxyl radical (HO^•) is known as the most reactive species of these. HO^• is generally produced from hydroperoxides through the Fenton reaction using reducing agents and metallic ions able to oscillate between different valencies (e.g. Cr²⁺, Mg²⁺, Fe²⁺, Ce²⁺). The various ROS react with different substrates: for example, HO^• reacts rapidly with all types of cellular components, whereas reacts primarily with protein Fe-S centres and ¹O₂ is particularly reactive with conjugated double bonds as found in polyunsaturated fatty acids (PUFAs) and aromatic amino acids (Requena and Stadtman, 1999; Requena et al., 2001).

NO^• is a gas emitted by plants, with the rate of evolution increasing under conditions such as pathogen challenge or hypoxia. However, exactly how NO^• evolution relates to its bioactivity in planta remains to be established. NO^• has both aqueous and lipid solubility, but is relatively reactive and easily oxidized to other nitrogen oxides. It reacts with superoxide to form peroxynitrite, with other cellular components such as transition metals and haem-containing proteins and with thiol groups to form S-nitrosothiols (Requena and Stadtman, 1999; Requena et al., 2001).

Both ROS and RNS, being reactive molecules, oxidize all types of cellular components. A large number of biological molecules such as carbohydrates, unsaturated lipids, proteins and nucleic acids are the target of these reactive species, and the damage may occur in different ways in several sites of the molecule. Proteins are the most abundant cellular targets of the oxidative species, more than DNA and lipids, making up 68% of the oxidized molecules in the cell. Investigation of reactive species effects in a cell can be performed through the analysis of modified components which requires sophisticated techniques. In fact, since ROS/RNS are highly reactive and short-lived species that do not accumulate to significant levels, it is not possible to measure them directly; rather, one must measure either the accumulation of biomolecules or the exogenously added indicators that are modified by both. This means that they leave different footprints in the cell in the form of different oxidatively modified components. The choice of which assay to use is a compromise between ease, ability to collect real-time data, need for spatial information, and instrumentation available. At any rate, techniques capable of directly measuring ROS/RNS in vivo and tracking lipids, nucleic acids, and protein oxidation can augment these studies by providing, among other things, a spatial component to the localization of stress at the tissue, cellular, and subcellular level. Most knowledge has been acquired through classical biochemical approaches, specially designed for the identification of carbonyl groups in proteins. More generally, the techniques developed for the identification of the oxidative stress-induced modifications on proteins often use immunological detection of the modified residues themselves (e.g. nitrotyrosine) or of haptens that can be selectively coupled to the modified residues (e.g. detection of carbonyls via immunodetection of coupled dinitrophenylhydrazine) (Levine et al., 1994). However, all of these assays are prone to numerous artefacts resulting from sample preparation and storage or from the analytical method itself, and all are limited in their ability to differentiate between different ROS molecules (for a review, see Halliwell and Whiteman, 2004). Moreover, focusing on a single modification does not render the complexity of the cellular response to oxidative stress, or redox protein regulation. The cellular response to oxidative stress, in fact, is not a passive one that merely results in the simple accumulation of modified proteins, so a classical approach to the phenomenon based on limited hypotheses will not reflect the complexity of the cell response, which involves a variety of changes in protein levels, controlled post-translational modifications, and oxidative damage to proteins (Rabilloud et al., 2005).

During the last decade it has become apparent that proteomics is the best-suited approach to the problem. Recent advances in mass spectrometry analytical techniques have already provided more accurate and more quantitative ways to measure oxidative modifications in the cell. An emerging branch of proteomics, the so-called ‘redox proteomics’, is not only determined simply by protein levels but also by post-translational modifications to proteins, which might be significant, particularly in cells undergoing an oxidative or nitrosative stress. Fortunately, several significant developments have been made in the field of redox proteomics to facilitate the determination of alterations to proteins under conditions of stress. Because alterations in the redox status of proteins are typically critical in regulating a protein's function, the identification of these proteins and the type of modifications present are of significant interest (for reviews see Ströher and Dietz, 2006; Møller et al., 2007).

In the vegetable kingdom ROS production and ROS-induced damage occur during biotic and abiotic stress, but ROS are associated with signalling as well (Apel and Hirt, 2004; Mittler et al., 2004; Foyer and Noctor, 2005; Gechev et al., 2006). Typically, more damage is observed during stress conditions in which ROS levels are also increased. At the same time the oxidized products can be important secondary signalling molecules, and in such cases damage and signalling are two sides of the same story, which can either coexist or cause severe imbalances (Fig. 1). This has led to the concept of ‘oxidative signalling’ instead of ‘oxidative stress’ (Foyer and Noctor, 2005). Some oxidative/nitrosative protein modifications, in fact, being specific and reversible, may play a key role in normal cellular physiology. The use of ROS as signalling molecules by plant cells suggests that, during the course of evolution, plants were able to achieve a high degree of control over ROS toxicity and are now using ROS as signalling molecules to control more specialized processes such as plant growth and defence, hormonal signalling, and development. Thus, the stress really occurs only when the oxidative species overwhelm the antioxidant defences of the cell.

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Total contribution of protein modifications to oxidative status in plant cells. When the presence of ROS and RNS species is limited, protein modifications mean intracellular communication and eventually the start of a real oxidative stress response. A ROS and RNS excess, not effectively countered by scavenger species, causes protein carbonylations, the formation of reactive groups, backbone cleavage, protein unfolding, and loss of functionality. A severe imbalance in ROS concentration inside the cellular environment leads to an alteration of cellular function and even to cellular death.

 
The purpose of this review is to present an overview of the latest research endeavours in the field of plant redox proteomics, with emphasis on those advancements being made in the field of free radical biology, to identify the role of post-translational modifications of proteins in developmental cell stresses, without losing sight of ROS signalling. To assist the reader as you plunge into the complexities of redox proteomics, the current knowledge about ROS and RNS turnover in plant cells is briefly summarized first, then the type and extent of oxidative modifications found in plant cell protein side chains are considered and, finally, proteomic technology used for the molecular identification of such modifications is reviewed.

	Production of reactive oxygen and nitrogen species in plants

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
ROS generation
Although plants may share some common traits in their responses to the various untoward environmental factors they may encounter, ROS production occurs via different mechanisms depending on whether the stress factor concerned is biotic or abiotic. However, under normal physiological activity, ROS in plants are continuously produced as by-products in a number of cellular compartments (Foyer and Harbinson, 1994), as a result of reactions involved in metabolic pathways. Under steady-state physiological conditions, antioxidative defences intervene to scavenge ROS and avoid harmful effects, often isolating them in specific cellular compartments (Alscher et al., 1997). However, several adverse environmental factors may modify the equilibrium existing between ROS production and the scavenging activity. As a result of these disturbances, intracellular levels of ROS may rise rapidly (Elstner, 1991; Tsugane et al., 1999) in a phenomenon known as ‘oxidative burst’ (Apostol et al., 1989). Plants can also generate an oxidative burst by activating various oxidases and peroxidases that produce ROS in response to certain environmental changes (Doke, 1985; Allan and Fluhr, 1997; Bolwell et al., 1998, 2002; Schopfer et al., 2001).

In plants there are three main types of ROS-producers acting at different cell loci: (i) electron-transport chains (ETC) in chloroplasts and mitochondria; (ii) some peroxidases and oxidases (NADPH oxidase, NADH oxidase, xanthine oxidase, lipoxygenase, glycolate oxidase, amine oxidase, etc.); (iii) photosensitizers such as chlorophyll molecules (Dat et al., 2000; Blokhina et al., 2003).

Electron-transport chains (ETC):
Respiration has special features in plant cells. While mitochondrial electron transport in most animals is based on a linear succession of redox reactions, the respiratory chain of plant mitochondria is branched at several points. Besides the ‘classical’ complexes I (NADH dehydrogenase), II (succinate dehydrogenase), III (cytochrome c redutase) and IV (cytochrome c oxidase), plant mitochondria contain at least five additional so-called ‘alternative’ oxidoreductases, which participate in respiratory electron transport (Fig. 2A). Four of these enzymes catalyse electron transfer from NADH or NADPH to ubiquinone and are termed ‘rotenone insensitive NADH dehydrogenases’. The fifth enzyme is a terminal oxidase called ‘alternative oxidase’ (AOX). It catalyses direct electron transfer from ubiquinol to molecular oxygen (Møller, 2001). All these alternative oxidoreductases do not couple electron transport to proton translocation across the inner mitochondrial membrane and therefore, seem to catalyse energy-wasting reactions. However, it is believed that these reactions are important, possibly because they are the basis for overflow-protection mechanisms of the respiratory chain in plant cells under certain physiological conditions (Møller, 2001). The main sites of ROS production in the mitochondrial ETC are complexes I and III, where the superoxide anion is formed and, in turn, is reduced by dismutation to H₂O₂ (Fig. 2A). H₂O₂, a compound of relatively low toxicity, can react with reduced Fe²⁺ and Cu⁺ to produce highly toxic hydroxyl radicals and, being uncharged, can also penetrate membranes and leave the mitochondrion (Sweetlove and Foyer, 2004). Whereas in mammals mitochondria are the main source of ROS (Halliwell and Gutteridge, 1989), in photosynthesizing plants their relative contribution to ROS production is very low (Purvis, 1997). In fact, in chloroplasts, light-driven photosynthetic processes are the main contributors to ROS production (Edreva, 2005). Normally, the electron flow from the excited photosystem centres is directed to NADP which is reduced to NADPH. It then enters the Calvin cycle and reduces the final electron acceptor, CO₂ (Fig. 2B). The processes catalysed by rubisco (carboxylase-oxygenase) both produce and consume O₂. If the ETC is overloaded, working under O₂-rich conditions, the leakage of electrons leads to an inevitable yield of ROS. Moreover, chlorophyll molecules, being endowed with photosensitive properties, mediate ROS generation after the input of light energy. In particular, a part of the electron flow is diverted from ferredoxin to O₂, reducing it to the superoxide free radical (Mehler, 1951).

View larger version (53K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. Schematic representation of the ROS production in the inner membrane of plant mitochondria (A) and the thylakoid membrane of chloroplasts (B). A. In addition to the four standard protein complexes found in nearly all other mitochondria, the ETC of plant mitochondria contains five additional enzymes (depicted in green). is formed at the levels of complex I and complex III. Arrows indicate the transport of electrons. AOX, alternative oxidase; ND, rotenone-insensitive dehydrogenase; Fum., fumarate; Succ., succinate; UQ, ubiquinone. B. Reduction of oxygen on the acceptor side of PSI, as a result of the photosynthetic transport of electrons, leads to the formation of superoxide anion, which can be further converted to H2O2 and HO•. Transfer of excitation energy from excited chlorophylls to oxygen in the light-harvesting complexes leads to the formation of 1O2. The production of ROS is enhanced by strong light and also by deceleration of the Calvin cycle. Arrows indicate the transport of electrons, whereas the dashed arrow indicates the transfer of excitation energy. Fd, ferredoxin; FNR, ferredoxin-NADP+ reductase; OEC, oxygen evolving complex; PC, plastocyanin.

 
The generation of through O₂ reduction is a rate-limiting step. Once produced, may follow different pathways: (i) on the internal, ‘lumen’ membrane surface (acidified upon illumination) may be protonated to which initiates lipid peroxidation; (ii) on the external, ‘stromal’ membrane surface is enzymatically (by SOD) or spontaneously dismutated to H₂O₂ and O₂. After the dismutation, H₂O₂ may be transformed through the Fenton reaction into the much more dangerous HO^•, at Fe-S centres where Fe²⁺ ions are available (Fig. 2B). Otherwise, H₂O₂ can be scavenged by catalases or the enzymes and metabolites of the ascorbate–glutathione cycle (Noctor and Foyer, 1998; Dat et al., 2000).

Chlorophyll sensitizers as a source of ROS:
Oxygen can be activated by light-dependent (photodynamic) reactions (classified as type I and type II) mediated by chlorophyll excitation (Hippeli et al., 1999). A photodynamic reaction undergoing charge separation in the excited pigment is referred to as reaction type I:

P (pigment)+lightP* (excited pigment)

P*₊P^– (charge separation)

₊P^–+O_{2 +}P+ (superoxide formation)

In the reaction type II chlorophyll is activated by energy transfer:

P (pigment)+lightP* (excited pigment)

P*+O₂¹O₂+P (input of excitation energy from P*+O₂ and generation of singlet oxygen)

The sequence of submolecular events leading to ¹O₂ production in chloroplasts is presented shortly in Fig. 3.

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. Generation of singlet oxygen through interaction of oxygen with triplet-state chlorophyll. Absorption of light energy causes one electron to be ejected from an electron pair in the chlorophyll molecule in the ground state (S0) to a highly energetic state (S2). When this electron loses energy, it drops down to lower energetic states, S1 and then T*. The transfer from S1 to T* (triplet) state is accompanied by a reversal of the electron spin. This is the key event generating the excited, triplet-state chlorophyll. Transfer of energy from it to the ground state oxygen produces spin reversal of one electron in O2, i.e. the formation of singlet oxygen (1O2), whereas the chlorophyll electron returns to its ground state S0. Chl, chlorophyll. (This figure is available in colour at JXB online.)

 
NO^• and RNS generation
NO^• is formed non-enzymatically in plants during normal cell metabolism both in the presence of ascorbate and as a by-product in nitrate reductase (NR) activities under hypoxic conditions; enzymatically, it is formed from nitrite by the activity of nitrite:nitric oxide reductase (Ni-NOR), and from L-arginine and oxygen by putative nitric oxide synthase (NOS). However, it is well known that plants under biotic or abiotic stress produce early and transient bursts of nitric oxide (NO^•). How many endogenous sources of NO^• exist is still unknown. The effects of NO^• may be harmful or beneficial depending on concentrations and environment (Fig. 4). At low concentrations, NO^• acts as a chemical messenger of the defences against stress and pathogens, integrating and differentiating time-dependent responses. As messenger, NO^• is produced in the picomolar or nanomolar range and the primary targets of NO^• are the haems, carriers of the three most important gases in plant metabolism (NO^•, CO₂, O₂), but also the iron-sulphur proteins. NO^• and thiols are nitrosylated to S-nitrosothiols, stored, and probably shuttled by the trans-nitrosylation of proteins. At high concentrations and in the presence of ROS, NO^• reacts to give rise to reactive nitrogen species (RNS) and in so doing creates oxidative and nitrosative stress. In particular, NO^• reacts with superoxide forming peroxynitrite and peroxynitrous acid. Peroxynitrite decomposes rapidly generating hydroxyl (HO^•) and nitrogen dioxide (). The latter initiates lipid peroxidation, oxidizes sulphhydryls, and nitrates the aromatic residues of proteins (Fig. 4). is considered an inhibitor of catalase and ascorbate peroxidase, enzymes reputed to be involved in the control of ROS (for a comprehensive review on nitric oxide and reactive nitrogen oxide species in plants, see Durzan and Pedroso, 2002).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. Diagram showing the production of NO• and RNS, with their effects on biological targets. At high concentrations, NO• reacts mainly with oxygen superoxide forming peroxynitrite (ONOO–) and peroxynitrous acid (ONOOH). In this way, NO• is intimately linked with ROS. Moreover, the reaction of NO• with O2 leads to the formation of the highly poisonous nitrogen dioxide (), dinitrogen tetroxide (N2O4), or both. At low concentrations, the direct effects of NO• predominate (dashed arrow) and haems and redox metals at iron–sulphur centres in proteins are the main targets. Ni-NOR, nitrite:nitric oxide reductase; Ni, nitrite reductase; NOS, nitric oxide synthase; NR, nitrate reductase; RSNOs, S-nitrosothiols.

 

	Topological distribution of ROS and NO• production in plant tissues

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
Despite the growing knowledge on the role of NO^• and ROS in signalling and cellular response to stress, less attention has been paid to the spatial distribution of these molecules in plant cells. Consequently, little information is available on the transport of ROS and NO^• from the site of origin to the place of action or detoxification. Recently, specific aquaporins have been shown to be able to facilitate the transmembrane movement of hydrogen peroxide (Bienert et al., 2007). On the other hand, whereas H₂O₂, ¹O₂, and NO^• can cross cell membranes, superoxides and hydroxide radicals cannot, so such information is useful in localizing where ROS is produced and oxidative stress is experienced. To this end, information on the contribution of different types of cells to the accumulation of ROS/RNS would increase our knowledge about the cellular response in plants to adverse conditions. Moreover, the tools for studying protein oxidation and protein turnover may uncover new mechanisms for regulating protein activities. At present, real-time measures of ROS and oxidative stress are limited and there are no truly non-invasive methods. However, recent developments in analytical methods, especially electron spin resonance and mass spectrometry, already provide more accurate and quantitative ways to measure ROS in the cell. In addition, fluorescent probes in combination with imaging techniques such as confocal laser scanning microscopy (Hideg, 2008; Sandalio et al., 2008), can offer both temporal and spatial information about ROS distribution, making it possible to monitor oxidative stress at the cellular or subcellular level.

	Oxidative/nitrosative protein modifications

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
The most reactive oxygen species in biology is the HO^• radical, which has a constant oxidation rate for proteins which is comparable to the rate of diffusion. It leads to non-specific protein oxidation, whereas all other ROS, having lower oxidation rates, react more specifically (Davies, 2005). With regard to NO^•, the most important physiological factors concerning oxidation are the sites and spatial distribution of its formation, flux, and the duration of its production. In fact, at NO^• concentrations below 1 µM and distant from the site of production, the direct effects of NO^• predominate, whereas at sites where high and sustained amounts of NO^• are produced, indirect effects prevail. They arise from interactions of NO^• with oxygen and superoxide, giving reactive nitrogen oxide species, among which the protonated form of peroxynitrite (ONOOH) is the most reactive product (Halliwell and Gutteridge, 2007).

Protein oxidation chemistry has been well-reviewed elsewhere (Davies, 2005), therefore in this review only a selection of the most noteworthy modifications will be reported with emphasis on the physiological and pathological effects of oxidized derivatives.

Sulphur-containing residues: cysteines and methionines
Among amino acids, the most oxidation-susceptible residues are the sulphur-containing ones, cysteine and methionine. The thiol of cysteine can be oxidized to a disulphide (PSSP), sulphenic acid (PSOH), sulphinic acid (PSO₂H) or sulphonic acid (PSO₃H) (Fig. 5A). The first two of these modifications are readily reversible, while the latter two are often described as irreversible. However, it is now emerging, that cysteine sulphinic acids are also reversible. This sulphinic acid switch has been demonstrated in peroxiredoxins, initially in micro-organisms (Biteau et al., 2003; Jacob et al., 2004) and more recently in mammalian and plant cells (Jeong et al., 2006; Rey et al., 2007). In particular, the 2-cysteine peroxiredoxins (2-Cys-Prxs) are known to be antioxidants that reduce peroxides through a thiol-based mechanism. During catalysis, these ubiquitous enzymes are occasionally inactivated by the substrate-dependent oxidation of the catalytic cysteine to the sulphinic acid (-SO₂H) form, and are reactivated by reduction by sulphiredoxin (Srx). In plants, 2-Cys-Prxs constitute the most abundant peroxiredoxins and are located in chloroplasts. Interestingly, recent data has established that, as in yeast and mammals, plant 2-Cys-Prxs are subject to substrate-mediated inactivation which is reversed by Srx, and the data suggests that the 2-Cys-Prx redox status and sulphiredoxin are parts of a signalling mechanism participating in plant responses to oxidative stress (Rey et al., 2007).

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. Commonly observed oxidative modifications of protein amino acids (A) cysteine; (B) methionine; (C) tyrosine; (D) tryptophan. All the amino acids are schematically represented as part of a polypeptide chain. However, the names shown are those of free amino acids for convenience.

 
In general, cysteine oxidation can be induced by hydrogen peroxide, superoxide, or nitric oxide (NO^•) and its derivatives, as well as more highly reactive species such as hydroxyl radicals (HO^•). Disulphide bonds may form intramolecularly within one protein, or intermolecularly between proteins (Fig. 5A); often the mechanism involves the initial formation of a sulphenic acid, which then reacts with a thiol to yield the disulphide. Mixed disulphides with small molecules such as cysteine (thiolation) or glutathione (glutathionylation) are also common (Fig. 5A), and it is thought that the formation of protein disulphides can protect the protein from further, potentially more damaging, oxidation. S-glutathionylation can occur in several ways (Klatt and Lamas, 2000; Giustarini et al., 2004; Ghezzi, 2005; Jacob et al., 2006): (i) reaction between protein thiols and intermediate S-nitrosothiols, such as S-nitrosoglutathione (GSNO) which is able to modify PSH by both protein S-nitrosation and S-glutathionylation; (ii) direct interaction between partially oxidized (activated) protein sulphhydryls, i.e. thiyl radical (RS^•, which can be formed by reaction with a hydroxyl radical), sulphenic acid, or protein S-nitrosothiol (S-nitrosated protein) and GSH; (iii) thiol/disulphide exchange reactions between protein thiols and GSSG or PSSG; (iv) direct interaction between a free protein cysteinyl residue and GSH triggered by many oxidants. Furthermore, glutathione sulphenic acid (GSOH) and glutathione disulphide S-monoxide [GS(O)SG] are considered alternative mediators of S-glutathionylation.

The messenger NO^• and its derivatives can covalently attach to a cysteine thiol to produce disulphides, sulphenic acid, sulphinic acid, sulphonic acid, and S-nitrosothiol (Fig. 5A). NO^• does not cause S-nitrosylation on its own, but it seems to involve S-nitrosothiols. Peroxynitrite can cause not only depletion of SH groups and other antioxidants, but also the oxidation of lipids, deamination of DNA bases, nitration of aromatic amino acid residues in proteins, as well as the oxidation of methionine to its sulphoxide. However, because of their reactivity with intracellular reducing agents, for example, ascorbic acid or glutathione (GSH), and with reduced metal ions, especially Cu⁺, nitrosothiols are exceptionally labile. This instability results in tissue half-lives of seconds to a few minutes and therefore provides a very sensitive mechanism for regulating cellular processes. S-nitrosylation is emerging as an important mechanism for the transduction of NO^• bioactivity. S-nitrosylation, in fact, potentially regulates the function of the protein: the majority of all NO^•-affected proteins seem to be regulated by S-nitrosylation of a single critical Cys residue, which occurs via oxygen-dependent chemical reactions or by the transfer of NO^• from a nitrosothiol to a protein sulphhydryl group (trans-nitrosylation). Growing evidence suggests, in fact, that nitric oxide is a central molecule in several physiological functions of the plant, ranging from development to defence responses. In a proteomic study, cell suspension cultures or leaves from Arabidopsis thaliana were treated with an NO^•-donor or gaseous NO^•, respectively, to generate S-nitrosothiols, subsequently S-nitrosylated proteins were detected using the biotin-switch method (see below). Biotin-labelled proteins were purified and analysed using nano-liquid chromatography in combination with mass spectrometry. Sixty-three proteins from cell cultures and 52 proteins from leaves were identified, confirming the existence of targets for S-nitrosylation in A. thaliana. Among these were stress-related, redox-related, signalling/regulating, structural, photosynthetic, and metabolic proteins, indicating that NO^• is involved in the regulation of all of these processes (Lindermayr et al., 2005). However, one of the best characterized functions of NO^• refers to its role in plant defence against pathogen attack, in particular in the establishment of the hypersensitive reactions (HR). A key step toward elucidating the mechanisms through which NO^• functions during the HR is the identification of the proteins that are subjected to this post-translational modification. Using a proteomic approach involving 2-DE and MS, Romero-Puertas et al. (2008) characterized, for the first time, changes in S-nitrosylated proteins in A. thaliana undergoing hypersensitive reactions. The 16 S-nitrosylated proteins identified were mostly enzymes serving intermediary metabolism, signalling, and antioxidant defence. Two proteins involved in the antioxidant machinery, monodehydroascorbate reductase (MDHAR) and a type II peroxiredoxin (PrxII E), were of particular interest because at the onset of the hypersensitive reactions the relative rates of NO^• and ROS (particularly and H₂O₂) are critical in channelling NO^• through the cell death program. The authors investigated the molecular mechanism for S-nitrosylation of PrxII E and found that it inhibits both the peroxidase and the peroxynitrite reductase activities of PrxII E, thereby revealing a novel regulatory mechanism for peroxiredoxins (Romero-Puertas et al., 2007). This suggests that NO^• may regulate the effects of its own radicals through S-nitrosylation of crucial components of the antioxidant defence system that function as common triggers for ROS and NO^•-mediated signalling events.

Like cysteine, methionine is also one of the most readily oxidized amino acids, owing to the presence of sulphur, and is susceptible to attack by most reactive oxygen or nitrogen species (Vogt, 1995). Oxidation of methionine usually yields methionine sulphoxide (R-SOCH₃, abbreviated as MetSO), a relatively commonly detected oxidative modification in biological systems, although a more severe attack can result in the formation of the sulphone (R-SO₂CH₃) (Fig. 5B). Methionine sulphoxide formation can be reversed by chemical reduction or by the action of methionine sulphoxide reductases, whereas methionine sulphone is thought to be irreversible and damaging to protein. In many proteins, oxidation of a few surface methionines has little effect on protein structure and function, but accumulation of methionine damage can cause changes in protein hydrophobicity and conformation, with concomitant effects on protein function. However, the ready enzymatic regeneration of methionine sulphoxide has led to the suggestion that methionine acts as an antioxidant to protect other amino acid residues from more deleterious damage (Levine et al., 1996). An example of the reversible oxidation of methionine to methionine sulphoxide occurs in the small heat shock protein of chloroplasts. This protein is inactivated by methionine sulphoxidation, and reactivated by reduction catalysed by the enzyme peptide methionine sulphoxide reductase using thioredoxin (Trx) as the reductant (Gustavsson et al., 2002). In A. thaliana, a null mutation in a gene encoding a cytosolic isoform of the enzyme showed increased ROS content, lipid peroxidation, and protein oxidation at the end of a long night, which was clearly stressful to the plant (Bechtold et al., 2004). It has been suggested that some peripheral methionine residues act as endogenous antioxidants protecting the active site and other sensitive domains in the protein while helping to remove ROS (Levine et al., 1996). It is quite likely that reversible methionine sulphoxidation will turn out to be an important regulatory mechanism (Sundby et al., 2005).

Tyrosine
Tyrosine oxidation can alter residue hydrophobicity, as may any changes in (sterically large) aromatic residues, with consequent effects on protein structure. Tyrosine phosphorylation in signal transduction makes tyrosine oxidation especially important, and since oxidation products of aromatic residues are even stable under acid hydrolysis they are useful biomarkers of protein oxidation. Tyrosine residues are converted to the 3,4-dihydroxy (dopa) derivative and also to bi-tyrosine cross-linked derivatives (Fig. 5C) which are formed by free radical attack, particularly metal-catalysed attack. Tyrosine has also been shown to be both nitrated and chlorinated in vivo by substitution with the electrophiles NO^• (from nitric oxide or peroxynitrite) or Cl⁺ (from HOCl), with nitration having been tentatively implicated in signalling. Addition of NO^• initially gives rise to nitrosotyrosine, which is rapidly and readily oxidized further in the presence of oxygen to nitrotyrosine (Fig. 5C). Tyrosyl radicals can also be directly nitrated, as in ribonucleotide reductase and photosystem II (Davis et al., 2001).

Tryptophan
Tryptophan oxidation is another apparently irreversible protein modification (Shacter, 2000). The formation of N-formylkynurenine by dioxygenation of tryptophan (Fig. 5D) was detected by two-dimensional gel electrophoresis and mass spectrometric analysis in numerous peptides from rice leaf and potato tuber mitochondria (Møller and Kristensen, 2006). Growing evidence suggests that Trp oxidation is not a random process. It is possible that exposed Trp residues act as intramolecular antioxidants, as suggested for Met residues (Levine et al., 1996). In fact, with the exception of the oxidation-sensitive aconitase (a Krebs cycle enzyme that contains an Fe-S centre), all of these modified proteins were either redox active or subunits in redox-active enzyme complexes (including the ETC). The same site was modified in (i) several adjacent spots containing the P-protein of the glycine decarboxylase complex, (ii) two different isoforms of the mitochondrial processing peptidase in complex III, and (iii) the same Trp residues in Mn-SOD in both rice and potato mitochondria (Møller and Kristensen, 2006). In the photosynthetic apparatus, two interesting cases of tryptophan oxidation were reported to be occurring in different PSII chlorophyll-binding proteins. The intrinsic PSII subunit named CP43 functions in light harvesting and plays a role in PSII assembly and structure. Interestingly, tandem mass spectrometry showed that the indole side chain of CP43 Trp-352 is post-translationally modified to kynurenine (+4 Da), a keto-/amino-/hydroxyl- (+16 Da) derivative, and a dihydro-hydroxy- (+18 Da) derivative of the indole side chain (Anderson et al., 2002). Peptide synthesis and MS/MS measurements confirmed the kynurenine assignment, where the +16 and +18 tryptophan modifications were postulated to be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine (Anderson et al., 2002). Interestingly, the authors hypothesized that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications play a role in signalling the turnover of the PSII reaction centre (Anderson et al., 2002). Along the same lines Rinalducci et al. (2005) reported the replacement of the tryptophan residue in the sequence ¹²⁷FGEAVWFK¹³⁴ of the Lhcb1 protein with N-formylkynurenine, as a consequence of ROS produced during thylakoid illumination.

Protein carbonylation
After the reactions involving the sulphur-containing amino acids, carbonylation is the most commonly occurring oxidative protein modification. Lysine, arginine, proline, and threonine side-chains can be oxidatively converted to reactive aldehyde or ketone groups (carbonylation) causing inactivation, crosslinking or breakdown of proteins (Levine and Stadtman, 2001; Dalle-Donne et al., 2003; Davies, 2005). A number of carbonylated proteins have been identified in the mitochondrial matrix, 20 of which were probably carbonylated in vivo and a further 31 were oxidized by an in vitro treatment with HO^• (Kristensen et al., 2004). There are no indications that carbonylation is reversible (Shacter, 2000). It has been reported that in A. thaliana protein carbonylation in total protein extracts increased during the vegetative phase, but decreased sharply at the start of the reproductive phase, staying relatively low until and during senescence. It was suggested that a low degree of protein carbonylation during the reproductive phase could be part of a strategy to limit the transfer of oxidatively damaged components to the offspring (Johansonn et al., 2004). In wheat leaves, protein carbonylation was higher in the mitochondria than in chloroplasts and peroxisomes (Bartoli et al., 2004). This indicates that the mitochondria are more susceptible to oxidative damage and/or the removal of modified proteins is less efficient in the mitochondria. In chloroplasts, three PSII subunits (CP47, D2, and D1) contain reactive groups that covalently bind amines and phenylhydrazine. It has been proposed that these reactive groups are carbonyl-containing, co- or post-translationally modified amino acids (Ouellette et al., 1998; Anderson et al., 2000). The identification of modified amino acid residues in one of the PSII subunits (CP47), was recently achieved using tandem mass spectrometry (Anderson et al., 2004). Modified residues were affinity-tagged with either biotin-LC-hydrazide or biocytin hydrazide, which are known to label carbonyl groups. The affinity-tagged subunit was isolated by denaturing gel electrophoresis, and tryptic peptides were then subjected to affinity purification and tandem mass spectrometry. By this procedure the authors found that the aspartic acid D348 of the CP47 subunit is post- or co-translationally modified to give a novel amino acid side chain, aspartyl aldehyde (Anderson et al., 2004).

	Proteomic approaches for the molecular characterization of oxidatively/nitrosatively modified proteins

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
Our knowledge of post-translational modifications induced by oxidative stress is far from complete and new oxidative modifications are likely to be discovered. Unfortunately, not all the proteomic approaches are capable of dealing properly with the specific features of the oxidative stress response. Oxidative stress is a constitutive phenomenon and the differences we expect to observe are mostly quantitative; moreover post-translational changes are expected to be a major component of the oxidative stress, thus the proteomic approach chosen must be able to deal with such quantitative changes as well as these complex post-translational modifications. Furthermore, there are no biochemical tools able to address all known oxidative modifications (for example, there are no antibodies against the oxidation products of methionine, tyrosine or tryptophan). These constraints rule out certain aspects of proteomics, such as shotgun approaches (Washburn et al., 2001) or isotope-coded, peptide-based methods (Gygi et al., 1999). To date, most of the proteomic studies of the oxidative stress response have used 2D electrophoresis as a protein separation and quantification tool, coupled with mass spectrometry (MS) as a protein characterization tool. This technique is able to separate proteins through quite small variations in the isoelectric point (pI), and many post-translational modifications can be detected in this way. The most typical example is phosphorylation, but some stages of cysteine oxidation (cysteine sulphinic and sulphonic acids) are also expected to induce pI changes. Last, but certainly not least, the separation power of 2D gels considerably simplifies the subsequent analysis by MS. The digestion of each spot on a 2D will give a few dozen peptides, producing a level of complexity that is easily managed by peptide fragment fingerprinting approaches based on matrix-assisted laser desorption/ionization (MALDI)-MS/MS, or nano-electrospray liquid chromatography (LC)-MS/MS approaches. This low complexity affords important sequence coverage. This implies that the likelihood of finding one of the few modified peptides in a protein altered by oxidative stress is higher with this technical set-up than with other strategies that manage more complex peptide mixtures, for example, shotgun approaches (for general considerations, see the review by Rabilloud et al., 2005). However, another aspect has to be considered. A whole tissue extract contains the products of ten of thousands of genes, each one of which can be modified by many post-translational processes. That will create a large number of electrophoretically discrete subpopulations of that protein. Thus, a given spot on the gel could easily contain many different proteins and the point is that only a fraction of them will be present in an amount sufficient to give a MS identification. At any rate, MS/MS approaches often allow both the description and the localization of the modification in the modified peptide. This statement does not mean, however, that such an assignment is trivial. As a rule of thumb, most modifications cause the MS signal to decrease considerably. For example, sulphation and cysteine oxidation in sulphinic or sulphonic acid alter the charge of the peptide, often making it negative. This decreases the ionization efficiency of peptides in cations, which is the usual and optimal ionization mode. In other cases (e.g. carbonylation) the modification favours peptide–peptide interactions (e.g. by Schiff base formation), which in turn decrease the peptide extraction yields and thus the signal. For these reasons, modification assignment is not easy and is poorly documented, except for phosphorylation, for which dedicated strategies have been described (Areces et al., 2004; Reinders and Sickmann, 2005).

Strategies to analyse oxidized/nitrosated cysteine
Identification of thiol modifications by gel-based labelling technologies:
Thiol modifications include direct oxidation, formation of mixed disulphides (e.g. with GSH, cysteine, and homocysteine), and the formation of intra/intermolecular protein disulphides (for the latter two processes see below) (Davies, 2005; Eaton, 2006). Thiol oxidation is of interest both as a consequence of oxidative stress and in increasingly recognized redox signalling. Protein thiols do not react with oxidants at their biological concentrations (Eaton, 2006). However, thiol pK_a values can be lowered by their surrounding environment which makes some cysteines particularly redox-sensitive. Protein -SH groups can be detected by radiolabelling with [³⁵S] and chemical tagging. Incubating cells in [³⁵S]cysteine results in labelled proteins, which may be visualized in autoradiograms of 2D SDS-PAGE separations (Westbrook et al., 2001). Low abundance radiolabelled proteins may co-migrate with abundant non-labelled proteins and peptides from the latter would dominate MS spectra. Even proteins lacking thiols altogether may be bound to [³⁵S] labelled proteins and again be mis-identified (Eaton, 2006). Therefore, identification of cysteine-containing peptides with a mass shift equivalent to [³⁵S] is good evidence of the identification of redox-modified proteins. Oxidized variants of -SH generally do not react with thiol-specific reagents such as maleimides iodoacetic acid, iodoacetamide (IAA), thiosulphates, and others. Thus it is possible to compare samples using labels covalently attached to these chemical groups. Identification of cysteinyl groups involved in disulphide bridge formation within the proteins was achieved by successive labelling with two different alkylating reagents of distinct molecular mass, namely IAA and 4-vinylpyridine (Maeda et al., 2005). It was shown that this approach does indeed allow verification of the redox state of specific cysteines in a complex protein mixture when the method was applied to the Trx-regulated barley -amylase/subtilisin inhibitor. After reduction of the disulphide bond with Trx, the newly accessible thiol groups were labelled with IAA (57 Da mass increase). A second reduction reaction with DTT reduced all remaining disulphide bonds that were labelled with 4-vinylpyridine (105 Da mass increase). After 2D gel electrophoresis, tryptic digestion of the interesting spots and MALDI-TOF analysis, the Trx accessible cysteines were identified by the peptide mass shifts of the untreated and DTT-reduced protein, due to the molecular mass difference between the differentially labelled cysteines (Maeda et al., 2005).

Usefulness of diagonal gel for studying disulphide bonds:
Diagonal gel electrophoresis is a form of two-dimensional analysis useful for investigating the subunit composition of multi-subunit proteins containing interchain disulphide bonds. Proteins are electrophoresed in the first dimension in a slab or tube gel under non-reducing conditions. The proteins are then reduced in the gel and this piece of gel is layered onto a second gel and electrophoresed at 90° to the original direction (Samelson, 2001) (Fig. 6). In the second gel, the proteins migrate at right angles to their original, first-dimension migration. The majority of cellular proteins are not disulphide-linked and will fall on the ‘diagonal’ in this system; that is, they migrate approximately equal distances in both directions during electrophoresis and lie approximately on the diagonal line connecting opposite corners of the gel. Upon reduction, component subunits of proteins connected by interchain disulphide bonds will resolve below the diagonal because the individual subunits migrate faster than the disulphide-linked complex during the second electrophoresis. By contrast, spots above the diagonal represents proteins with intramolecular disulphide bridges (Fig. 6). In fact proteins with intramolecular bonds often have a more compact structure; they will be unfolded following reduction and usually migrate more slowly in the second dimension (Samelson, 2001). Diagonal gel separations can be used to identify redox-based modifications in a similar way to 2D SDS-PAGE since disulphide bridged variants are usually highly resolved. As early as 1979, Anderson and Manabe applied this method to plant protein extracts to investigate whether dithiol-disulphide proteins are present in the thylakoid membrane. However, due to a shortage of sensitive analytical tools, the identity of the studied proteins remained obscure at that time. Recently, the development of efficient high throughput mass spectrometry and the accessibility of genome-based protein databases have allowed a revival of the method of diagonal SDS-PAGE. Yano et al. (2001b) combined two-dimensional separation with a thioredoxin reduction system in order to identify targets of a specific dithiol/disulphide exchange system in seeds. The same authors correlated the mobilization of the seed storage proteins with their reduction state (Yano et al., 2001a). Mobilization was followed by application of non-reducing/reducing two-dimensional-PAGE after labelling of the sulphhydryl groups of seed proteins with the thiol monobromobimane (mBBR) reagent. The work showed that storage proteins, particularly the glutenins, were reduced during germination. Therefore, the authors suggested that limited proteolysis in the early stages of seed germination following disulphide reduction enables the proteolytic enzymes to digest the intermediates rapidly.

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6. Separation of redox-active proteins through diagonal 2D-SDS-PAGE. The prominent diagonal across the gel contains the proteins without redox-active cysteines. On the contrary, proteins with an intermolecular disulphide bridge (homo- or heterodimers) migrate below the diagonal, whereas proteins with intramolecular bridges are resolved above the diagonal line. (This figure is available in colour at JXB online.)

 
In combination with Western blot analysis for sensitive immunodetection, Holtzapffel et al. (2003) used this approach to identify redox-active alternative oxidase proteins from tomato fruit mitochondria. In a recent report (Winger et al., 2007) this diagonal approach was used more systematically in Arabidopsis mitochondria complementing the thiol-tagging approach that Balmer et al. (2004) used in spinach, pea, and potato, and the work by Rouhier et al. (2005) in poplar. Interestingly, comparison of the protein sequences of the identified proteins with homologues from other species has identified specific Cys residues that may be responsible for plant-specific redox modulations of mitochondrial proteins (Winger et al., 2007).

COmbined FRActional DIagonal Chromatography (COFRADIC):
Similar to diagonal gel electrophoresis, the COFRADIC method has been developed to reduce the high complexity of proteome-derived peptide mixtures and is based on a specific modification reaction between the two subsequent runs on the same reverse-phase column which alters the retention time of specific peptides (for a review, see Gevaert et al., 2006). In the original publication the procedure was developed for sorting methionyl peptides (Gevaert et al., 2002), but a somewhat different sorting strategy led to the isolation of cysteinyl peptides. Here, proteins were first reduced and then reacted with Ellman's reagent (5,5-dithiobis[2-nitrobenzoic acid] or DTNB), making a heterodisulphide bridge between the thiol group of cysteine and a hydrophobic nitrobenzoic acid group. After the first chromatographic run, the hydrophobic TNB group was released from the complex by reduction. The thiol-containing proteins became more hydrophilic and their retention time was shortened. As a consequence, the proteins of interest eluted 3–10 min prior to their elution time in the first run. Afterwards, LC-MS/MS analysis can provide information about the identity of the proteins. To date, the method has been used for sorting cysteinyl peptides in analysing the proteome of human plasma and human platelets (Gevaert et al., 2004). However, given COFRADIC's intrinsic versatility, the technique also opens up interesting perspectives for future developments in the characterization of in vivo protein processing and post-translational modifications in plant tissues.

Isotope-coded affinity tags (ICAT):
This technique uses a certain type of marker which consists of three different parts: (i) a thiol-reactive compound (an iodoacetamide analogue), (ii) a linker containing either heavy or light isotopes, and (iii) a biotin tag for separation by avidin-coupled affinity chromatography. Only free cysteines are accessible for these markers. Therefore, isotope-coded affinity tags were originally developed to compare complex protein mixtures (Gygi et al., 1999) and applied in redox proteomics. After exposing equivalent protein samples to either control conditions or hydrogen peroxide, they are differentially labelled with the heavy or light form of the ICAT. The protein samples are mixed and, after tryptic digestion, the labelled peptides are separated by affinity chromatography. The captured peptides are analysed by LC-MS. Because oxidized cysteines are not susceptible to labelling with ICAT, the labelling intensity decreases from the control to the H₂O₂ sample (Sethuraman et al., 2004).

Investigation of S-glutathionylation:
A number of methods are available for the study of protein S-glutathiolation in cells, tissues, and in vitro models of oxidative stress that have recently been reviewed in detail (Ghezzi and Bonetto, 2003). Metabolic labelling of the GSH pool with radioactive [³⁵S] has been extensively utilized over several decades and has also been coupled to modern proteomic technology to allow identification of proteins that are S-glutathiolated during oxidative stress (Fratelli et al., 2002). S-Glutathiolation alters the charge of a protein, which facilitates the detection of oxidized proteins using Western immunoblotting of samples separated by IEF gel electrophoresis (Ghezzi et al., 2001). S-Glutathiolation induces IEF gel shifts that are reversed by treatment with reducing agents such as DTT. However, the application of this method is limited because it can only be used to study known targets of S-glutathiolation, the protein also has to be amenable to separation by IEF, and an antibody to it has to be available. Some authors have used N-biotinyl analogues of reduced cysteine, GSH, or GSH ethyl ester in the study of protein S-thiolation (Sullivan et al., 2000; Eaton et al., 2002a, b, c, 2003; Ito et al., 2003). However, methods based on labelled GSH have several drawbacks. Only proteins that are present in high quantities can be identified, furthermore a protein that was extensively glutathionylated under basal (non-stressed) conditions may not incorporate labelled GSH further because it is already fully oxidized. The total redox-responsive disulphide proteome of A. thaliana has recently been investigated using large-scale proteomic techniques (Lee et al., 2004). However, few studies have focused specifically on the ability of proteins to form mixed protein disulphides with GSH and how this modification may regulate their activity. Ito et al. (2003) fed a biotinylated glutathione ester to Arabidopsis cell cultures and reported that some 20 proteins underwent thiolation, although only two of these polypeptides were subsequently identified by sequencing, highlighting the difficulties of working in vivo. An efficient in vitro method systematically to identify Arabidopsis polypeptides that are capable of undergoing rapid S-glutathionylation has recently been published (Dixon et al., 2005). After labelling the intracellular glutathione pool with [³⁵S]cysteine, suspension cultures of Arabidopsis were shown to undergo a large increase in protein thiolation following treatment with the oxidant tert-butylhydroperoxide. To identify proteins undergoing thiolation, a combination of in vivo and in vitro labelling methods utilizing biotinylated, oxidized glutathione (GSSG-biotin) was developed to isolate Arabidopsis proteins/protein complexes that can be reversibly glutathionylated. Following two-dimensional polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry proteomics, a total of 79 polypeptides were identified, representing a mixture of proteins that underwent direct thiolation, as well as proteins complexed with thiolated polypeptides (Dixon et al., 2005).

Methods for detection of S-nitrosylation:
Protein S-nitrosylation is a labile post-translational modification, not stable enough to survive the rigours of denaturing SDS-polyacrylamide gel electrophoresis. Thus, while anti-S-nitrosocysteine antibodies have been generated and applied in immunohistochemistry studies, they are of little use in the context of proteomics investigations. Recently, the Biotin Switch method was established to allow detection of protein S-nitrosylation after SDS-polyacrylamide gel electrophoresis and electroblotting (Jaffrey and Snyder, 2001). The procedure allows specific biotinylation of S-nitrosylated cysteine residues in proteins. First, free thiols are blocked by incubation with a thiol-specific methiolating agent (methyl methanethiosulphonate or MMTS) in the presence of sodium dodecyl sulphate to ensure access to buried cysteine residues. Conversely, the methiolating agent does not react with nitrosothiols or disulphide bonds under the reaction conditions used. Excess of MMTS is removed using a spin column or by acetone precipitation. Next, nitrosothiol bonds are selectively decomposed to thiols with ascorbate. Finally, the free thiol groups are reacted with the sulphhydryl-specific biotinylation reagent, N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) (Fig. 7). The labelled proteins may then be separated by SDS-polyacrylamide gel electrophoresis, electroblotted, and detected by standard immunoblotting methods using anti-biotin antibodies or streptavidin. Labelled proteins may be selectively enriched using streptavidin affinity chromatography as well (Sell et al., 2008). The Biotin Switch method has permitted new proteomic investigations into the fundamental role of protein S-nitrosylation in cellular signalling (Jaffrey and Snyder, 2001).

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7. Scheme of the biotin switch method. A hypothetical protein is indicated with cysteines in either the free thiol, disulphide, or nitrosothiol conformations. In the first step, free thiols are blocked using MMTS. Next, nitrosylated cysteine residues are selectively reduced with ascorbate and the newly generated free thiols are finally S-biotinylated with biotin-HPDP. The biotinylated proteins can be detected directly by Western blotting with antibodies specific for biotin or using avidin or streptavidin. Antibodies can be radiolabelled, fluorescently or enzymatically labelled, as is known in the art. Additionally, tagged proteins can also be isolated from affinity columns or beads. PSH, protein sulphhydryl groups;. PSNO, S-nitrosated proteins.

 
Recently, modifications of the biotin switch method have emerged. The SNO site identification was developed as a proteomic approach that enables simultaneous identification of SNO cysteine sites and their cognate proteins in complex biological mixtures (Hao et al., 2006). The biotinylated proteins were tryptic digested and the resulting peptides which contain target cysteines for S-nitrosylation were purified by pull down using Neutravidin and identified by mass spectrometry.

Another related technique is the so-called His-tag switch method that comprises first the blocking of free thiols by N-ethylmaleimide (NEM) alkylation, secondly the SNO specific reducing step with ascorbate, and finally the addition of a his-tag by alkylation for detection (Camerini et al., 2007). This strategy allows the purification and the unambiguous identification of the modified cysteines by mass spectrometry.

Analysis of oxidized/nitrosated tyrosine:
Nitration of protein tyrosine residues leading to o-nitrotyrosine is a most intriguing post-translational modification. Nitration is a relatively stable modification that can be suitably analysed by different specific techniques, such as immunochemical techniques (using anti-o-nitrotyrosine antibodies), HPLC in combination with various detection systems, and GC/MS (Söderling et al., 2003). The chromatographic methods usually involve analysis of o-nitrotyrosine released by acid or enzymatic hydrolysis of protein extracts (Kanski and Schöneich, 2005). A combination of MS techniques has been used to identify the specific tyrosine residues nitrated in vitro in model proteins (Ducrocq et al., 1998; Curcuruto et al., 1999). More recently, a higher throughput characterization of protein targets for tyrosine nitration in cells and several tissues has been attempted by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), partial transfer onto poly(vinylidene difluoride) membranes, and Western blot analysis with anti-nitrotyrosine antibody to identify the modified proteins (Kanski et al., 2003, 2005). Alignment of the Western blots with the 2D-PAGE gels enables identification of immunopositive protein spots. These are then excised, trypsin digested, and identified by either peptide mass fingerprinting procedures using MALDI mass spectrometry or capillary LC-MS/MS analyses (Aulak et al., 2004). Usually, the direct mass spectrometric identification of nitrated peptides is not difficult and merely relies on the correct alignment of Western blot analyses and 2D gels. However, a prerequisite for successful identification is the presence of a single protein per gel spot. If the gel spot contains multiple proteins or protein isoforms (Molloy, 2000), unambiguous identification of the nitrated protein can only be obtained by tandem MS sequencing of the o-nitrotyrosine-containing peptide.

Recently Amoresano et al. (2006) developed a novel approach to label phospho-Ser/-Thr residues in proteins selectively using dansyl modification coupled with tandem mass spectrometry experiments in precursor ion/MS³ scan mode, taking advantage of a hybrid mass spectrometer. This concept led to the development of a general method, reporter ion generation tag (RIGhT), of potential interest for large-scale proteomic identification of a broad range of PTMs. In a subsequent paper, the same authors reported the extension of this innovative strategy to the selective isolation and identification of o-nitrotyrosine-containing proteins (Amoresano et al., 2007). The methodology was first tested on in vitro nitrated BSA as a model protein and then applied to more complex matrices. However, there is no evidence of similar studies on vegetal samples.

Analysis of carbonylation:
Carbonylation occurs mainly via the HO^• radical and this has been suggested to be rather non-specific (Levine and Stadtman, 2001). However, recent studies have found evidence of selective protein carbonylation (McDonagh et al., 2005).

Protein carbonyls react quantitatively with hydrazines to form hydrazones (Levine et al., 1990). After 2D SDS-PAGE, proteins can be transferred to nitrocellulose membranes and carbonylated proteins labelled with hydrazine–dinitrophenol (DNP) followed by detection with anti-DNP (Nakamura and Goto, 1996). ELISA assays have been developed for the quantitation of DNPH-derivatized carbonyls. The same chemistry can be used in combination with gel electrophoresis, followed by immunodetection. Alternatively, Yoo and Regnier (2004) have developed a biotinylation strategy for the specific labelling of carbonylated proteins after 2-DE. Another possible strategy is to use DNPH derivatization in combination with anti-DNPH antibodies to immunoprecipitate and enrich carbonylated proteins, which has been demonstrated by England and Cotter (2004) in the study of ER protein susceptibility to oxidation by 2-DE and MALDI-TOF MS, and by Kristensen et al. (2004) in a rice leaf mitochondrial oxidation study with 2-D LC-MS/MS. An alternative is to use affinity baits for the specific isolation of carbonylated proteins. So far, these innovative methods have been applied to standard proteins and yeast lysate. For example, one can use biotin hydrazine for the derivatization of ketones and aldehydes, and avidin columns for the specific isolation of derivatized peptides and proteins (Mirzaei and Regnier, 2005, 2006, 2007). Interestingly, Mirzaei and Regnier (2007) compared three different strategies based on biotin hydrazine tagging of carbonyls, affinity selection, proteolysis, RP-HPLC, and MS, and found that performing the affinity selection and chromatography at the protein level before proteolysis and mass spectrometric protein identification, was more informative. This is because working with intact protein allowed the detection of crosslinked or truncated proteins. Using a similar approach, Roe et al. (2007) directly derivatized glass beads with a hydrazine group, allowing spin down isolation of carbonylated proteins.

Table 1 summarizes the methodological approaches described in this review with the attempt to guide the reader through the various techniques.

View this table: [in this window] [in a new window]   Table 1. List of the most utilized methods in redox proteomics

 

	Identifications of ROS/RNS damage in plants

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
It is well established that oxidation of proteins by radicals, in the presence of O₂, can result in major alterations to the physical and chemical nature of the target, with these changes including not only oxidation of side-chain groups, but also changes in hydrophobicity and conformation, formation of new reactive groups, altered susceptibility to proteolytic enzymes, unfolding, backbone fragmentation and cross-linking (Davies, 1987; Dean et al., 1997; Stadtman and Levine, 2003). These alterations can result in the loss of structural or enzymatic activity of the protein and hence biological perturbations. Backbone cleavage is believed to occur via the formation of carbon-centred radicals at the -carbon position. Reaction of these species with molecular oxygen gives rise to peroxyl radicals, which can subsequently react via a number of different pathways to produce backbone cleavage (Davies, 1996; Headlam and Davies, 2002).

Several case studies of backbone degradation in plants as a consequence of interactions between ROS and proteins are outlined here.

Ribulose-1,5-bisphosphate carboxylase/oxygenase
Rubisco is located in the stroma of chloroplasts where it catalyses the primary reactions of CO₂ assimilation and photorespiration. ROS trigger Rubisco degradation and the large subunit of Rubisco is site-specifically cleaved into five major fragments in light-treated leaf discs under chilling temperatures (Nakano et al., 2006). In this case, the fragmentation was completely inhibited by n-propyl gallate or 1,2-dihydrobenzene-3,5-disulphonic acid, suggesting the involvement of HO^• and , respectively. Transition metal ions like Fe²⁺ and Cu⁺ bound to proteins can generate ROS that can potentially cleave the polypeptide backbone. The ROS generated probably only diffuse relatively short distances from the generation site, suggesting that proximity is a very important parameter for the cleavage reaction of proteins (Luo et al., 2002).

Photosystem I
PSI is degraded in intact leaves under chilling-light conditions, resulting in the destruction of the Fe-S centres and detectable degradation of the PSI-A and PSI-B reaction centre subunits (Allen, 1995; Sonoike et al., 1995, 1997; Rajagopal et al., 2005). Apart from PSI-A and PSI-B, other smaller polypeptides of PSI were degraded during strong light illumination, such as PSI-C, PSI-D, and PSI-E that comprise the stromal side of photosystem I (Rajagopal et al., 2005). The data indicate the involvement of ROS, probably HO^• produced by the Fenton reaction between photoreduced Fe-S centres and H₂O₂. PSI photodestruction results in the release of free iron from the damaged Fe-S centres. Iron ions leaking from the thylakoid membrane into the stroma of the chloroplast could lead to the generation of HO^•, which can attack stromal proteins such as Rubisco (for a review of PSI photoinhibition and repair, see Scheller and Haldrup, 2005). Recently, in a report using photosystem I submembrane fractions illuminated with strong light (2000 µmol m^–2 s^–1 photosynthetically active radiation), damage to the light-harvesting complex was reported and the photodegradation profile of each polypeptide was analysed. At the light intensity used, all PSI antenna proteins were affected, although to different extents. In particular, immunoblot analysis showed that Lhca2 is the most sensitive whereas Lhca4 is the most stable subunit (Rajagopal et al., 2005).

D1 protein (PSII)
Under illumination, D1 has the highest turnover rate of all thylakoid membrane proteins. The cause of D1 fragmentation is likely to be ¹O₂ produced by the reaction centre chlorophyll of PSII (P680), because in vitro treatment with ¹O₂-generating substances results in a similar fragmentation pattern of the D1 protein, as observed under in vivo photoinhibitory conditions (Okada et al., 1996). However, it is not the ¹O₂, per se, that causes cleavage of the peptide backbone, because the fragmentation is not observed in a mutant lacking an FtsH protease. Rather, the accumulation of oxidized amino acids in the D1 protein triggers a conformational change that renders the D1 protein susceptible to the FtsH protease (Silva et al., 2003).

Light-harvesting complex
Evidence for light-mediated degradation of isolated light-harvesting proteins from photosystem II and involvement of oxygen free radicals in the process was reported by Zolla and Rinalducci (2002). Using mass spectrometry and amino acid sequencing, it was revealed that the primary cleavages take place in the hydrophilic portion of the NH₂ region where oxygen-containing radicals attack randomly and not at specific sites. Interestingly, electron spin resonance measurements showed that protein damage starts with the generation of ¹O₂, presumably from a triplet chlorophyll, acting as a Type II photosensitizer which directly attacks the amino acids causing the proteins to degrade completely into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H₂O₂ were not involved initially in the reaction mechanism (Rinalducci et al., 2004).

Other thylakoid proteins
Using a global approach based on proteomics Rinalducci et al. (2005) demonstrated the involvement of ROS in inducing truncated forms and high-molecular aggregates in photosynthetic proteins when thylakoids are exposed to visible light. Consistent with this, 2D gel electrophoresis of spinach thylakoid membranes revealed different spot profiles when performed in complete darkness compared with the illumination of the sample with low-intensity visible light. In particular, a large number of new spots with lower molecular masses were detected, and their identification by MS/MS revealed that most of them are simply native proteins that have been truncated. As well as those proteins known to be targeted by ROS (such as D1, LHCII) several other polypeptides belonging to different complexes appeared to be damaged. A new spot containing a truncated form of the OEC23 protein was revealed and the sequence coverage obtained suggested a possible removal of some amino acids at the N-terminal portion. Breakdown products of the ATP synthase -chain were also detected, as well as new fragments deriving from CP47 and cyt b₅₅₉ (Rinalducci et al., 2005).

	Concluding remarks

Top Abstract Introduction Production of reactive oxygen... Topological distribution of ROS... Oxidative/nitrosative protein... Proteomic approaches for the... Identifications of ROS/RNS... Concluding remarks References

 
Previously, oxidative modification of proteins was thought to represent a detrimental process in which the modified proteins were irreversibly inactivated, leading to cellular dysfunction. While this is still the case in many situations, it is now clear that oxidative/nitrosative protein modifications can be specific and reversible and, thus, may play a key role in normal cellular physiology. However, central to this issue is the question which remains unresolved: does the formation of oxidized proteins have a significant, direct physiological or pathological impact, or is it a secondary phenomenon? A clear delineation of the causal connections cannot be given at present, but a growing body of evidence indicates that high levels of ROS/RNS induce distinct pathological cell consequences that greatly amplify and propagate injury, leading to irreversible cell and tissue degeneration, supporting the hypothesis that ROS/RNS-induced protein modification may be of physiological significance, and that in some cellular stresses it is not solely a secondary consequence. In this sense, we believe that redox proteomics will have a central role in the definition of redox molecular mechanisms associated with plant stresses. On the other hand, proteins are involved in virtually every cellular function, therefore the proteome dictates the functional phenotype in each tissue or organ. This phenotype varies continuously under normal conditions and can change as a result of oxidative/nitrosative stress, contributing to initiation and/or progression of cellular defence or adaptation. For specific proteins, stress-induced modifications will substantially affect function, which in turn has the potential to affect other proteins. The result is a dynamic, ongoing process of protein expression and modification. Thus, the challenge for future studies will be to incorporate this knowledge into a framework whereby these complex functional and regulatory alterations in the cellular proteome can be used to increase our understanding of and improve the treatment of plants under stress.

As described in this review, the challenge now facing this research field is to sort out which protein markers or combinations of markers are predictive of cellular alteration associated with oxidative/nitrosative stress. The opportunities for identification of proteins involved during the perturbation strictly linked to oxidative/nitrosative stress are clear and compelling. Recent advances in proteomics technologies have now made it possible to examine critically the molecular targets within the mitochondrion and chloroplasts that contribute to cellular dysfunction. While a tremendous amount of progress has been made in this area in recent years, it should be pointed out that there are still deficiencies in these techniques and approaches that greatly impede these analyses. On the one hand, progress in the immunochemistry field is hampered by the fact that only a small number of modifications can be detected in proteins due to the limited number of antibodies that are available to detect these modifications. On the other hand, even though significant advancements have been made in instrumentation, mass spectrometry is still not sufficiently sensitive to detect, quantify, and localize these post-translational modifications, particularly when these alterations may encompass only a few amino acids within the protein. Thus, quantification of the degree of modification within a protein remains a challenge. However, even with these limitations, cutting edge research in the field has now begun to identify specific molecular modifications to selective amino acids within cellular proteins that occur from exposure to ROS, RNS, and electrophilic lipids under both normal and stressed-physiological conditions. Furthermore, comparison of the molecular fingerprints obtained in cells with those produced by various in vitro oxidation/nitrosation/nitration systems will indicate those biochemical pathways creating damage in vivo. All these data will decipher the potential roles played by ROS/RNS-induced modifications in stressed cells. In this sense, redox proteomics is opening new scenarios to gain insight into molecular mechanisms involved in the stress-induced cell. New proteomic tools, in the near future, will facilitate the identification of protein markers still unknown, allowing their complete characterization in a given stressed-physiological condition. Future progress in genomics, metabolomics, proteomics, and systems biology will result in more studies on global cellular responses to oxidative stress on transcript, protein, and metabolite levels, providing data for mathematical modelling of the biochemical networks involved.

These findings will help us to establish relationships between the cellular hallmarks of a particular stress and the functional and/or structural alterations to proteins.

	Acknowledgements

 
This work was financially supported by the Italian Ministry for University and Research (MIUR-PRIN 2006) and by National Blood Centre (ISS).

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