Advertisement

Detection, identification, and quantification of oxidative protein modifications

Open AccessPublished:October 31, 2019DOI:https://doi.org/10.1074/jbc.REV119.006217
      Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively-modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications—at the amino acid, peptide, or protein level—and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes.

      Introduction

      Biological systems are exposed to a wide variety of oxidizing species—both free radicals and two-electron oxidants. These species are often termed “reactive oxygen species,” although this is a misleading term, as the reactivity of these species varies enormously (see below). Oxidants are generated both deliberately (e.g. to kill invading pathogens or as intermediates in enzymatic reactions) or unintentionally (e.g. via electron leakage from electron transport chains, metabolism of drugs, exposure to chemicals, pollutants, and radiation). These processes have been reviewed in Refs.
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      .
      The formation of these oxidants and their reactions are limited by cellular and organismal defense systems, which include enzymes that remove oxidants or oxidant precursors (e.g. superoxide dismutases, peroxiredoxins, thioredoxin/thioredoxin reductase, GSH peroxidase isoforms, and catalases), and water- and lipid-soluble oxidant scavengers, including thiols (e.g. GSH and thioredoxin), ascorbic acid (vitamin C), urate, tocopherol isoforms (vitamin E), quinols (e.g. reduced coenzyme Q10), carotenoids, and polyphenols. Although these systems are efficient, and in many cases show redundancy (i.e. multiple processes remove the same species), they are not 100% effective, and a large body of data indicates that biological targets suffer resulting damage. These protective systems are therefore complemented by systems that either repair damage or remove the modified molecules (e.g. methionine sulfoxide reductases, disulfide reductases/isomerases, glutaredoxins, sulfiredoxins, proteasomes, lysosomes, proteases, phospholipases, and DNA repair enzymes) (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ).
      Despite this battery of preventative and repair systems, many studies have reported increased damage, and accumulation of this, in human, animal, and microbial and plant systems exposed to stress conditions (reviewed in Refs.
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). A higher level of damage may arise from increased oxidant generation, a decrease or failure of defense systems, or (most commonly) a mixture of both processes, as many defense systems are themselves subject to damage or show reduced activity due to co-factor depletion. This concept of an altered balance between formation and removal gave rise to the term “oxidative stress” (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ), although it is now apparent that this is an oversimplification of a complex picture, as limited stress (“eustress”) can be beneficial in priming and protecting a system against greater damage (“distress”) (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). Increasing age is often associated with a decrease in enzyme levels or activity, and in some cases decreased levels of co-factors and essential trace elements, such that increased levels of oxidants and modified products are formed (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). These changes can be accelerated by disease or environmental factors, despite the presence of feedback loops (e.g. antioxidant-response elements, including the Nrf-2 pathway; DNA damage–response element; OxyR; SoxRS) that up-regulate the synthesis of protective species (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). In this study, we review the basic chemistry and biochemistry of protein modification by oxidants, with a focus on methods available for the detection, identification, and quantification of these changes.

      Proteins as targets of oxidative damage

      Proteins are major components of most biological systems and constitute ~70% of the dry mass of cells and tissues. The rate of reaction of an oxidant with a biological target depends on the concentration of the target, multiplied by the rate constant for its reaction with the oxidant. Both of these factors result in proteins being major targets for damage as proteins are both present at high concentrations (up to 1–3 mm in plasma and 5–10 mm in cells) and have high rate constants for reaction with oxidants. Thus, oxidant damage in most biological systems is likely to be skewed toward protein modification (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). This is clearly an oversimplification of a complex situation, as other factors are known to play an important role, including localization of the generating system relative to the target and particularly membrane barriers, micro-environments, binding, or association of the oxidant system to a target, the occurrence of secondary reactions, and intra- and intermolecular transfer reactions (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). However, it is likely that proteins are major sites of damage in many situations, although it should also be noted that the extent of damage and its biological importance may be very different (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). Thus, low levels of modification of a critical target may have much greater consequences than a high level of damage to noncritical materials.
      Radicals (e.g. HO, CO3-., NO2·, ROO, RO, R, and many others), two-electron oxidants (e.g. peroxides, 1O2, O3, ONOOH, HOCl, and related species), and metal–oxo complexes can all modify proteins, although the reactivity and selectivity of these oxidants are highly variable (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). Reactions of secondary products (e.g. aldehydes, quinones, and dehydroalanine) are a further source of modifications (
      • Grimsrud P.A.
      • Xie H.
      • Griffin T.J.
      • Bernlohr D.A.
      Oxidative stress and covalent modification of protein with bioactive aldehydes.
      ,
      • Shu N.
      • Lorentzen L.G.
      • Davies M.J.
      Reaction of quinones with proteins: kinetics of adduct formation, effects on enzymatic activity and protein structure, and potential reversibility of modifications.
      ). Together, these generate a wide variety of post-translational modifications that alter amino acid and protein composition and structure, charge, hydrophobicity/hydrophilicity, folding, and function (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ,
      • Gianazza E.
      • Crawford J.
      • Miller I.
      Detecting oxidative post-translational modifications in proteins.
      ,
      • Dalle-Donne I.
      • Aldini G.
      • Carini M.
      • Colombo R.
      • Rossi R.
      • Milzani A.
      Protein carbonylation, cellular dysfunction, and disease progression.
      ).

      Nature and reactivity of oxidant species

      In the following section, a brief overview is presented on the formation and reactivity of a number of key oxidant species relevant to mammalian systems. Although each of these species is described as single entities, it should be noted that nearly all of these species undergo further interconversion reactions as illustrated in Fig. 1 (although this is situation-dependent) that result in complex mixtures in most reaction systems (
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ).
      Figure thumbnail gr1
      Figure 1Overview of interconversion processes of common biological oxidants. The extent of these reactions depends on the circumstances and reaction conditions and is therefore only intended as guide to the complexity of examining oxidant reactions. Adapted from Ref.
      • Davies M.J.
      Protein oxidation and peroxidation.
      .

      Superoxide radical-anions (O2-.)

      Superoxide O2-. radicals are generated continuously by most organisms as a result of the use of O2 as a terminal electron acceptor in some electron transport chains, such as those of mitochondria, the endoplasmic reticulum cytochrome P450 system, and the plasma membrane NADH/NADPH oxidase systems (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ). Incomplete coupling of electron transfer results in single electron leakage to O2 (estimated at 1–3%) (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ). O2-. is poorly reactive, with iron–sulfur complexes a significant target (
      • Hausladen A.
      • Fridovich I.
      Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not.
      ). O2-. undergoes rapid spontaneous, and superoxide dismutase-catalyzed, disproportionation to give hydrogen peroxide (H2O2) and O2, with the former being a precursor of further oxidizing species, as well as being a reactive species in its own right (
      • Fridovich I.
      Superoxide dismutases.
      ). O2-. is generated, at very high fluxes over short time periods, by membrane-associated NADPH oxidases (NOXs and DUOXs), at the expense of NADPH via an “oxidative burst” (
      • Babior B.M.
      The respiratory burst oxidase.
      ). O2-. is also formed by heme proteins (cytochromes, oxyhemoglobin, and oxymyoglobin) (
      • Misra H.P.
      • Fridovich I.
      The generation of superoxide radical during the autoxidation of hemoglobin.
      ), uncoupled nitric-oxide synthase, and xanthine oxidase, among others (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ).

      Hydrogen peroxide (H2O2)

      In addition to formation from O2-. by dismutation, H2O2 is also generated directly by a number of enzymes (e.g. NADPH oxidase-4, monoamine oxidases, hexose oxidases, amino acid oxidases, other oxidoreductases, protein-disulfide isomerases, and Ero1p during protein synthesis and folding (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      )). Direct oxidation of biological targets by H2O2 is both limited in extent and is usually slow. Thus, direct reaction is limited to Cys, selenocysteine, and Met residues with these typically occurring with very low rate constants (see also below), with the exception of reaction with some specialized enzymes (e.g. peroxiredoxins and GSH peroxidases), which have catalytic centers that facilitate rapid O–O bond cleavage. Such environments can elevate the rate constant for reaction by a million-fold (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ). H2O2 is a substrate for a large family of peroxidase enzymes, with these reactions used both synthetically (e.g. in the formation of thyroid hormones by thyroid peroxidase and in the generation of collagen matrices by peroxidasins) and to kill invading pathogens (
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.
      ).

      Hypohalous acids and other reactive halogen species

      Reaction of H2O2 with heme peroxidases, such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase, results in the formation of hypohalous acids (HOX, X = Cl, Br, I, or SCN) (
      • Davies M.J.
      • Hawkins C.L.
      • Pattison D.I.
      • Rees M.D.
      Mammalian heme peroxidases: from molecular mechanisms to health implications.
      ). These vary markedly in reactivity and oxidizing capacity, with hypochlorous acid (HOCl, familiar to many as the active component in household bleach) being the most reactive and powerful oxidant (
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      ,
      • Pattison D.I.
      • Davies M.J.
      • Hawkins C.L.
      Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids.
      ). HOCl (which exists in equilibrium with Cl2 at low pH values) is a key component of the innate immune response against pathogens, with this generated at high concentrations in neutrophil phagolysosomes (
      • Klebanoff S.J.
      • Kettle A.J.
      • Rosen H.
      • Winterbourn C.C.
      • Nauseef W.M.
      Myeloperoxidase: a front-line defender against phagocytosed microorganisms.
      ). Release of myeloperoxidase to the extracellular space (instead of into phagolysosomes) and subsequent reaction with H2O2 can, however, result in host tissue damage, with the level and activity of myeloperoxidase associated with tissue damage in acute and chronic inflammatory conditions (
      • Klebanoff S.J.
      • Kettle A.J.
      • Rosen H.
      • Winterbourn C.C.
      • Nauseef W.M.
      Myeloperoxidase: a front-line defender against phagocytosed microorganisms.
      ). Comparison of HOCl and H2O2 illustrates a key point about oxidants and their reactivity: H2O2 is a strong oxidant (reduction potential 1.32 V) and a more powerful oxidant than HOCl (reduction potential 1.28 V), but this reacts very slowly when compared with HOCl (cf. the rate constant, k, for reaction of HOCl with the amino acid methionine is ~108-fold higher than for H2O2 (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ,
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      )), so evolution has favored the use of HOCl over H2O2, for kinetic rather than thermodynamic reasons. The low reactivity of H2O2 is likely to be one of the major reasons why this species is used biologically as a messenger molecule: slow and selective reactivity allows for specificity in transmission of messages as only a limited number of select targets are likely to be activated. Highly-reactive species such as HOCl would not allow such specificity of transfer of information.

      Nitric oxide (NO)

      NO is a key vascular regulator and messenger molecule, with this generated from arginine by nitric-oxide synthase enzymes (NOSs)
      The abbreviations used are: NOS
      nitric-oxide synthase
      OPA
      o-phthaldialdehyde
      DMPO
      5-dimethyl-1-pyrroline N-oxide
      TNB
      as 5-thio-2-nitrobenzoic acid
      DTNB
      5,5′-dithiobis(2-nitrobenzoic acid
      AGE
      advanced glycation end product
      DOPA
      3,4-dihydroxyphenylalanine
      SEC
      size-exclusion chromatography
      FOX
      ferrous oxidation-xylenol orange
      UPLC
      ultra-performance liquid chromatography.
      (
      • Moncada S.
      • Palmer R.M.
      • Higgs E.A.
      Nitric oxide: physiology, pathophysiology, and pharmacology.
      ). The concentration generated by constitutive NOS isoforms is low (pico- to nanomolar), consistent with a regulatory function, but higher levels (up to micromolar) are formed by the inducible NOS isoform of macrophages, at sites of inflammation (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). NO reacts slowly with most biological targets, consistent with its role as a signaling molecule (
      • Moncada S.
      • Palmer R.M.
      • Higgs E.A.
      Nitric oxide: physiology, pathophysiology, and pharmacology.
      ,
      • Förstermann U.
      • Sessa W.C.
      Nitric oxide synthases: regulation and function.
      ,
      • Davis K.L.
      • Martin E.
      • Turko I.V.
      • Murad F.
      Novel effects of nitric oxide.
      ), with fast reactions occurring primarily with transition metal ions (e.g. the iron center of heme proteins, including that of soluble guanylate cyclase, the major effector of NO signaling) and with other radicals (
      • Moncada S.
      • Palmer R.M.
      • Higgs E.A.
      Nitric oxide: physiology, pathophysiology, and pharmacology.
      ,
      • Cary S.P.
      • Winger J.A.
      • Derbyshire E.R.
      • Marletta M.A.
      Nitric oxide signaling: no longer simply on or off.
      ). Reaction with other radicals can be protective (e.g. by terminating lipid peroxidation chain reactions) but also damaging when the product is a powerful oxidant, as is seen in the formation of peroxynitrous acid (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ).

      Peroxynitrous acid (ONOOH)

      Diffusion-controlled (i.e. k 109–1010 m−1 s−1) reaction of NO with O2-. gives ONOO(peroxynitrite). This species exists in equilibrium with the corresponding conjugate acid peroxynitrous acid (ONOOH), with the pKa (6.8) favoring the anion at most biological pH values (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). However, the acid (ONOOH) form is typically the more reactive species with protein targets. Reaction with CO2 and some boronic acid probes are exceptions, with the rate constant for these targets being higher with ONOO (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). The reactivity of ONOOH is therefore modulated by CO2 (which is in equilibrium with HCO3) as a result of the formation of ONOOCO2, which decomposes to give CO3-. and NO2· that can either diffuse out of the solvent cage or recombine (reviewed in Ref.
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). ONOOH is a potent oxidizing/nitrating species (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ) that can give rise to both two-electron and one-electron oxidation products. The former arises from direct reactions of ONOOH, and the latter is formed from limited homolysis to give HO and NO2·, and subsequent radical chemistry (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ).

      Hydroxyl (HO) and other oxygen-centered radicals

      Metal ion (primarily Fe and Cu)-catalyzed decomposition of H2O2 generates the highly-reactive and powerful oxidant HO via Fenton and pseudo-Fenton reactions (
      • Koppenol W.H.
      The centennial of the Fenton reaction.
      ); this species is also formed directly from water by high-energy radiation (
      • von Sonntag C.
      The Chemical Basis of Radiation Biology.
      ). Metal ion–catalyzed decomposition of organic and lipid hydroperoxides gives alkoxyl radicals (RO) that are less powerful oxidants than HO, but more reactive than peroxyl radicals (ROO) that are typically generated from rapid (typically diffusion-controlled) addition of O2 to carbon-centered radicals (R) (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). The latter arise from hydrogen atom abstraction from biological targets by reactive radicals (
      • von Sonntag C.
      The Chemical Basis of Radiation Biology.
      ). R and ROO are key intermediates in lipid peroxidation chain reactions, i.e. typically initiated by hydrogen abstraction from methylene groups of polyunsaturated fatty acids. ROO are the key chain carriers in lipid peroxidation (
      • Yin H.
      • Porter N.A.
      New insights regarding the autoxidation of polyunsaturated fatty acids.
      ), but ROO also appear to play a role in (short) chain reactions on proteins (
      • Neuzil J.
      • Gebicki J.M.
      • Stocker R.
      Radical-induced chain oxidation of proteins and its inhibition by chain-breaking antioxidants.
      ).

      UV light, singlet oxygen (1O2), and other photochemically-generated species

      UV light with very short wavelengths (UVC) is strongly absorbed by atmospheric molecules, such as ozone, and hence does not give rise to significant effects at the earth’s surface. In contrast, longer wavelength UV light, particularly UVB (λ 280–320 nm) and UVA (λ 320–400 nm) wavelengths, can oxidize molecules via light absorption by suitable chromophores and generation of either excited state species (i.e. species with an electron in a higher orbital, type 2 photoreactions) or radicals (type 1 photoreactions), as a result of photoejection of an electron (
      • Pattison D.I.
      • Rahmanto A.S.
      • Davies M.J.
      Photo-oxidation of proteins.
      ,
      • Davies M.J.
      Reactive species formed on proteins exposed to singlet oxygen.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ). The excited state species (usually triplet species due to the short lifetime of singlet states) can either induce direct oxidation by, for example, electron or hydrogen atom abstraction or by energy transfer to molecular oxygen O2 to give singlet oxygen (1O2) (
      • Pattison D.I.
      • Rahmanto A.S.
      • Davies M.J.
      Photo-oxidation of proteins.
      ,
      • Davies M.J.
      Reactive species formed on proteins exposed to singlet oxygen.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ). In addition to its formation by type 2 photoreactions, 1O2 is also formed by some enzyme-catalyzed reactions, via termination reactions of ROO, and some metal ion-catalyzed processes; 1O2 is therefore an important intermediate in both light-induced and “dark” reactions (
      • Davies M.J.
      Reactive species formed on proteins exposed to singlet oxygen.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ).

      Negative and positive aspects of oxidative damage

      Oxidant species can generate damage to all components of biological systems, including lipids, proteins, and DNA (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ), and these modifications have been linked to a wide range of pathologies, including some cancers, neurological disorders (Alzheimer’s and Parkinson’s diseases and amyotrophic lateral sclerosis), sepsis, hypertension, cardiovascular diseases, including atherosclerosis, ischemia-reperfusion injury to multiple organs, renal and ocular damage, cataracts, chronic obstructive pulmonary disease, cystic fibrosis, asthma, rheumatoid and osteoarthritis, motor neuron disease, irritable bowel syndrome, pancreatitis, hepatitis, sunburn and photodamage, and many more (
      • Halliwell B.
      • Gutteridge J.M.
      Free Radicals in Biology & Medicine.
      ,
      • Sies H.
      • Berndt C.
      • Jones D.P.
      Oxidative stress.
      ,
      • Butterfield D.A.
      • Perluigi M.
      • Reed T.
      • Muharib T.
      • Hughes C.P.
      • Robinson R.A.
      • Sultana R.
      Redox proteomics in selected neurodegenerative disorders: from its infancy to future applications.
      ). Although it is likely that some of these are only associations (i.e. oxidation is not causative but merely a consequence of other injurious processes), oxidative damage appears, at least in some cases, to be a contributing factor (i.e. causative). Methodologies that can detect and quantify oxidative damage are therefore of considerable importance, not least as potential biomarkers to assess therapeutic strategies. Oxidation can also be a valuable tool in the treatment of some diseases, with radiation and photodynamic therapy of tumors (
      • Symonds P.
      • Deehan C.
      • Meredith C.
      • Mills J.
      Walter and Miller's Textbook of Radiotherapy: Radiation Physics, Therapy and Oncology.
      ,
      • Kwiatkowski S.
      • Knap B.
      • Przystupski D.
      • Saczko J.
      • Keędzierska E.
      • Knap-Czop K.
      • Kotlińska J.
      • Michel O.
      • Kotowski K.
      • Kulbacka J.
      Photodynamic therapy- mechanisms, photosensitizers and combinations.
      ,
      • Beharry A.A.
      Next-generation photodynamic therapy: new probes for cancer imaging and treatment.
      ), the use of redox-cycling drugs (e.g. doxorubicin and nitroaromatics (
      • Tacar O.
      • Sriamornsak P.
      • Dass C.R.
      Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems.
      )), and photochemical tissue bonding in wound closure after surgery being good examples (
      • Xu N.
      • Yao M.
      • Farinelli W.
      • Hajjarian Z.
      • Wang Y.
      • Redmond R.W.
      • Kochevar I.E.
      Light-activated sealing of skin wounds.
      ). The following section outlines current knowledge of the modifications arising from oxidant interactions with proteins, and subsequently how these can be detected and quantified.

      Protein modifications induced by reactive oxidants

      The majority of radical reactions with proteins occur via three major pathways— hydrogen atom abstraction from C–H, S–H, N–H, or O–H bonds, electron abstraction from electron-rich sites, and addition to electron-rich centers (aromatic rings and sulfur species) (
      • Hawkins C.L.
      • Davies M.J.
      Generation and propagation of radical reactions on proteins.
      ). The first of these reactions results in the formation of carbon-centered species (R), thiyl radical (RS), nitrogen-centered species (primarily indolyl radicals from Trp), and oxygen-centered radicals (primarily phenoxyl radicals from Tyr). Most R radicals, including those generated from oxidation of aliphatic side chains (Leu, Ile, Val, and Pro, etc.) react rapidly with O2 to give ROO at diffusion-controlled rates (k ~109 m−1 s−1) (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). Although these reactions are fast, the O2 concentration is low in most biological samples (5–100 μm), which may limit ROO formation and result in dimers (R–R) when the R concentration is relatively high (Fig. 2) (
      • Dizdaroglu M.
      • Simic M.G.
      Isolation and characterization of radiation-induced aliphatic peptide dimers.
      ). In contrast, RS, Tyr phenoxyl radicals, and Trp indolyl radicals react with O2 with much lower rate constants (k <107 m−1 s−1 for RS, ≪105 m−1 s−1 for TrpN, and <103 m−1 s−1 for TyrO (
      • Schöneich C.
      Thiyl radicals and induction of protein degradation.
      ,
      • Fang X.
      • Jin F.
      • Jin H.
      • von Sonntag C.
      Reaction of the superoxide radical with the N-centered radical derived from N-acetyltryptophan methyl ester.
      ,
      • Hunter E.P.
      • Desrosiers M.F.
      • Simic M.G.
      The effect of oxygen, antioxidants, and superoxide radical on tyrosine phenoxyl radical dimerization.
      )), resulting in higher yields of cross-linked products, including disulfides (RSSR), Trp–Trp, and Tyr–Tyr (
      • Hägglund P.
      • Mariotti M.
      • Davies M.J.
      Identification and characterization of protein cross-links induced by oxidative reactions.
      ). Crossed dimers (e.g. Tyr–Trp) are also known (
      • Hägglund P.
      • Mariotti M.
      • Davies M.J.
      Identification and characterization of protein cross-links induced by oxidative reactions.
      ,
      • Leinisch F.
      • Mariotti M.
      • Rykaer M.
      • Lopez-Alarcon C.
      • Hägglund P.
      • Davies M.J.
      Peroxyl radical- and photo-oxidation of glucose-6-phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues.
      ). The chemical structures of some of the most abundant and commonly examined products are given in Fig. 3.
      Figure thumbnail gr2
      Figure 2Summary of O2-dependent reactions of carbon, peroxyl, and alkoxyl radical reactions on proteins and the occurrence of short-chain reactions. In this scheme, R, R′, and R″ are used to designate carbon-centered species with different chemical structures.
      Figure thumbnail gr3
      Figure 3Chemical structures of some of the most abundant and/or commonly examined side-chain oxidation products.
      For ROO generated on aliphatic side chains, hydrogen-atom abstraction is a major reaction with other X–H bonds, with this resulting in hydroperoxide (ROOH) formation (Fig. 2). These are major products with multiple different attacking species (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ,
      • Gebicki J.M.
      Protein hydroperoxides as new reactive oxygen species.
      ). Dimerization reactions of ROO have also been characterized, but these appear to be of modest importance, particularly when the radical flux is low (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). When this does occur, and the ROO is a tertiary species (e.g. on Val and Leu), a tetroxide is generated (ROOOOR) that can decompose to give two alkoxyl radicals (RO) and O2 (Fig. 2) (
      • Russell G.A.
      Deuterium-isotope effects in the autoxidation of aralkyl hydrocarbons. Mechanism of the interaction of peroxy radicals.
      ,
      ). The RO can subsequently undergo β-scission fragmentation reactions, to give a carbonyl compound and a further radical (Fig. 2) (
      • Headlam H.A.
      • Mortimer A.
      • Easton C.J.
      • Davies M.J.
      β-Scission of C-3 (β-carbon) alkoxyl radicals on peptides and proteins: a novel pathway which results in the formation of α-carbon radicals and the loss of amino acid side chains.
      ,
      • Headlam H.A.
      • Davies M.J.
      Markers of protein oxidation: different oxidants give rise to variable yields of bound and released carbonyl products.
      ). These processes may be partly responsible for the occurrence of (short) chain reactions on proteins and the generation of protein-bound (or released) carbonyls (
      • Headlam H.A.
      • Mortimer A.
      • Easton C.J.
      • Davies M.J.
      β-Scission of C-3 (β-carbon) alkoxyl radicals on peptides and proteins: a novel pathway which results in the formation of α-carbon radicals and the loss of amino acid side chains.
      ,
      • Headlam H.A.
      • Davies M.J.
      Markers of protein oxidation: different oxidants give rise to variable yields of bound and released carbonyl products.
      ). Alternatively, RO may undergo hydrogen atom abstraction radicals to generate alcohols (Fig. 2). With primary or secondary ROO, dimerization reactions yield 1 mol of alcohol, carbonyl, and O2 (Fig. 2) (
      • Russell G.A.
      Deuterium-isotope effects in the autoxidation of aralkyl hydrocarbons. Mechanism of the interaction of peroxy radicals.
      ). The major products from radical-mediated oxidation of aliphatic side chains are therefore hydroperoxides, alcohols, carbonyls, and fragmentation products.
      Highly-reactive radicals such as HO react with little selectivity (
      • Buxton G.V.
      • Greenstock C.L.
      • Helman W.P.
      • Ross A.B.
      Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms, and hydroxyl radicals (OH/O) in aqueous solution.
      ), except when these are generated in a site-specific manner, such as by bound metal ions, with this resulting in damage localized to the vicinity of the binding site (
      • Rykaer M.
      • Svensson B.
      • Davies M.J.
      • Hägglund P.
      Unrestricted mass spectrometric data analysis for identification, localization and quantification of oxidative protein modifications.
      ). Other less-reactive radicals can show marked selectivity. As most biologically-important radicals are electrophilic, they react most rapidly, and to the greatest extent, with electron-rich sites (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ) resulting in damage to a subset of amino acid side chains; these data are summarized in Table 1. These include the sulfur-containing amino acids Cys, Met, and cystine, and the aromatic residues, Trp, Tyr, Phe, and His. For both Cys and His, the reactions are pH-dependent, with the rate of oxidation occurring more rapidly at higher pH values (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). In the case of Cys, a wide range of products can be formed, including disulfides and oxyacids (
      • Poole L.B.
      The basics of thiols and cysteines in redox biology and chemistry.
      ,
      • Devarie-Baez N.O.
      • Silva Lopez E.I.
      • Furdui C.M.
      Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ). With Met, the major product is usually the sulfoxide (
      • Schöneich C.
      Methionine oxidation by reactive oxygen species: reaction mechanisms and relevance to Alzheimer’s disease.
      ), and to a much lesser extent the sulfone and carbon-centered radical products (
      • Schöneich C.
      Methionine oxidation by reactive oxygen species: reaction mechanisms and relevance to Alzheimer’s disease.
      ). For the aromatic amino acids, hydroxylated and dimeric species predominate, although with both Trp and His multiple ring opened products are generated (
      • Hawkins C.L.
      • Davies M.J.
      Generation and propagation of radical reactions on proteins.
      ,
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). A (nonexhaustive) list of products arising from such reactions is given in Table 2.
      Table 1Overview of selectivity of different oxidizing species for protein side chains
      Major targets
      Radical oxidants
       Hydroxyl radical: HOAll, including peptide backbone
       Alkoxyl radical: ROAll, but slower reactions than with HO
       Peroxyl radical: ROOCys, Met, Trp, Tyr
       Superoxide radical anion: O2-.Fe-S clusters
       Carbonate radical anion: CO3-.Cys, Met, Trp, Tyr, His
       Nitrogen dioxide radical: NO3-.Cys, Tyr, Trp (and Tyr/Trp radicals)
      Two-electron (non-radical) oxidants
       Peroxynitrous acid: ONOOHCys, cystine, Met, Trp, selenomethionine (Sec)
       UVB light (λ 280–320 nm)Trp, Tyr, cystine
       UVA light (λ 320–400 nm)No direct amino acid absorption
       Singlet oxygen: 1O2Cys, Met, Trp, Tyr, His, Sec
       Hypochlorous acid: HOClCys, Met, cystine, His, α-amino, Lys, Trp, Tyr (slow), Sec, selenomethionine, Arg (slow)
       Hypobromous acid: HOBrCys, Met, His, cystine, α-amino, Lys, Trp, Tyr, Sec
       Hypothiocyanous acid: HOSCNCys, Sec
       Hydrogen peroxide (H2O2) and other peroxidesCys, Sec, Met (slow), selenomethionine
       Quinones and aldehydesCys, Sec, Lys, Arg
      Table 2Selected major products (both stable and unstable) generated from oxidation of protein side chains
      Amino acid side chainThree-letter codeSingle-letter codeProducts
      AlanineAlaASerine
      Dehydroalanine
      Lanthionine (dehydroalanine–Cys cross-link)
      Lysinoalanine (dehydroalanine–Lys cross-link)
      Hydroperoxide
      Carbonyl (serine aldehyde, 2-oxo species)
      ArginineArgRHydroperoxides
      Alcohols
      Carbonyls (glutamic semi-aldehyde and others)
      His–Arg cross-links
      AsparagineAsnNHydroperoxides
      Aspartic acidAspDHydroperoxides
      Carbonyl
      Decarboxylated species (serine aldehyde)
      CysteineCysCCystine (disulfide)
      Sulfenic acid: RSOH
      Sulfinic acid: RSO2H
      Sulfonic acid: RSO3H
      Sulfenamide (sulfenyl amide): RS-NHR′
      Sulfinamide (sulfinyl amide): RSO-NHR′
      Sulfonamide (sulfonyl amide): RSO2-NHR′
      Nitrosocysteine: RSNO
      Nitrocysteine: RSNO2
      Sulfenylchloride: RSCl
      Persulfides: R(S)nH
      Multiple adducts to α-,β-unsaturated aldehydes, aldehydes, and quinones
      Lanthionine (dehydroalanine-Cys cross-link)
      His–Cys cross-link
      Thioethers (addition products)
      Glutamic acidGluEHydroperoxides
      Alcohols
      Carbonyls (3-oxo species)
      Decarboxylated species (aspartate semi-aldehyde)
      GlutamineGlnQHydroperoxides
      Carbonyls (3-oxo species)
      GlycineGlyGHydroperoxide
      HistidineHisHHydroperoxides and endoperoxides
      Hydroxyhistidine
      2-Oxohistidine
      Nitrohistidine
      Aspartyl urea (ring-opened product)
      Formyl asparagine (ring-opened product)
      Asparagine (ring-opened product)
      Aspartic acid (ring-opened product)
      Di-histidine cross-link (His–His)
      His–Cys cross-link
      His–Lys cross-link
      His–Arg cross-link
      IsoleucineIleIHydroperoxides
      Alcohols (hydroxyisoleucines)
      Carbonyls (3- and 4-oxo species)
      LeucineLeuLHydroperoxides
      Alcohols (hydroxyleucines)
      Carbonyls (4-oxo species)
      LysineLysKHydroperoxides
      Alcohols (hydroxylysines)
      Carbonyls (α-aminoadipic semi-aldehyde and others)
      Chloramines (RNHCl)
      Bromamines (RNHBr)
      Nitriles
      Lysinoalanine (dehydroalanine–Lys cross-link)
      MethionineMetMMethionine sulfoxide: RSOR′
      Methionine sulfone: RSO2R′
      Dehydromethionine
      Carbonyls (aspartate 4-semialdehyde arising from loss of–SMe function)
      Homocysteic acid: RSO3H (cleavage of S–CH3 bond)
      PhenylalaninePheF2-Hydroxyphenylalanine (ortho-Tyr)
      3-Hydroxyphenylalanine (meta-Tyr)
      Tyr (4-hydroxyphenylalanine)
      2- or 4-nitrophenylalanine
      ProlineProPHydroperoxides
      Alcohols (hydroxyprolines)
      Carbonyls (2-pyrrolidinone and ring-opened species such as glutamic semi-aldehyde)
      SelenocysteineSecUMixed seleno-thiol cross-linked species (RSe-SR′)
      Selenenic acid: RSeOH
      Seleninic acid: RSeO2H
      Selenonic acid: RSeO3H
      Dehydroalanine
      SelenomethionineSelenomethionine selenoxide
      SerineSerSCarbonyls (serine aldehyde, 2-oxo species)
      ThreonineThrTCarbonyls (2-amino-3-ketobutyric acid and others)
      TryptophanTrpWHydroperoxides and endoperoxides
      N-Formylkynurenine
      Hydroxy N-formylkynurenine
      Di-hydroxy N-formylkynurenine
      Kynurenine
      Kynurenic acid
      3-Hydroxykynurenine (and downstream products, including xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, and picolinic acid)
      Hydroxytryptophan (multiple isomers)
      5- and 6-nitrotryptophan
      Chlorotryptophan
      Hydropyrroloindole
      2-Oxindole species
      Di-oxindole species
      Hydroxytryptophandione
      Di-tryptophan (multiple isomers with both C–C and C–N linkages)
      Trp–Tyr cross-link species
      TyrosineTyrYHydroperoxides and endoperoxides
      DOPA
      DOPA quinone
      Trihydroxyphenylalanine (TOPA)
      3-Nitrotyrosine
      3,5-Dinitrotyrosine
      3-Chlorotyrosine
      3,5-Dichlorotyrosine
      3-Bromotyrosine
      3,5-Dibromotyrosine
      Di-tyrosine: Tyr–Tyr cross-link (both C–C and C–O linkages
      Trp–Tyr cross-link species
      ValineValVHydroperoxides
      Alcohols (3- and 4-hydroxyvalines)
      Carbonyls (3-oxo species)
      Radical-mediated damage can also be detected on the peptide backbone (
      • Hawkins C.L.
      • Davies M.J.
      Generation and propagation of radical reactions on proteins.
      ,
      • Garrison W.M.
      Current Topics in Radiation Research.
      ,
      • Davies M.J.
      Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage.
      ), with this appearing to occur primarily via hydrogen-atom abstraction from the α-carbon (the side-chain attachment site) (
      • Davies M.J.
      Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage.
      ,
      • Morgan P.E.
      • Pattison D.I.
      • Davies M.J.
      Quantification of hydroxyl radical-derived oxidation products in peptides containing glycine, alanine, valine, and proline.
      ). Subsequent reactions of the initial R formed in this process result in fragmentation of the peptide backbone, with this occurring via two different pathways involving ROO and RO (Fig. 4) (
      • Hawkins C.L.
      • Davies M.J.
      Generation and propagation of radical reactions on proteins.
      ,
      • Garrison W.M.
      Current Topics in Radiation Research.
      ,
      • Davies M.J.
      Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage.
      ). These pathways have been reviewed recently (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). With species such as HO, this can result in a large range of different cleavage sites along a protein backbone (often detected as a “smear” on protein gels), although some selectivity in fragmentation has been reported (
      • Schuessler H.
      • Davies J.V.
      Radiation-induced reduction reactions with bovine serum albumin.
      ,
      • Schuessler H.
      • Denkl P.
      X-ray inactivation of lactate dehydrogenase in dilute solution.
      ), particularly at metal ion–binding sites (
      • Chevion M.
      A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals.
      ). This may also be due to particular stabilizing factors, such as the capacity to form a planar intermediate that maximizes stabilization of the intermediate α-carbon species (
      • Easton C.J.
      • Hay M.P.
      Preferential reactivity of glycine residues in free radical reactions of amino acid derivatives.
      ) or a high level of solvent exposure on the surface of a protein (e.g. at turns between helices).
      Figure thumbnail gr4
      Figure 4Overview of radical reactions resulting in cleavage of the protein backbone. This can arise from both direct reactions at backbone sites (principally at the α-carbon) and also indirectly via initial oxidation at side-chain sites with subsequent radical transfer to the backbone, either intra- or intermolecularly. For further details see main text and Refs.
      • Davies M.J.
      The oxidative environment and protein damage.
      ,
      • Davies M.J.
      Protein oxidation and peroxidation.
      ,
      • Devarie-Baez N.O.
      • Silva Lopez E.I.
      • Furdui C.M.
      Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      .
      Two-electron oxidants typically show markedly greater selectivity than most radicals, due to the higher-energy barriers for many of these reactions (Table 1) (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). For species such as ONOOH, direct two-electron processes can occur (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ), including reaction with Cys, cystine, and Met residues to give oxygenated species (Table 1). Oxidation of metal-ion centers can also occur via two-electron pathways. However, these reactions occur in competition with homolysis of ONOOH to give radicals (and hence one-electron oxidation products), and reaction of the anion ONOO with CO2 to give the corresponding carbonate adduct (although the identity of this species is disputed (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • Radi R.
      Carbon dioxide-catalyzed peroxynitrite reactivity–The resilience of the radical mechanism after two decades of research.
      ,
      • Serrano-Luginbuehl S.
      • Kissner R.
      • Koppenol W.H.
      Reaction of CO2 with ONOO: one molecule of CO2 is not enough.
      )). The adduct has been reported to have a short life-time (a few nanoseconds (
      • Augusto O.
      • Goldstein S.
      • Hurst J.K.
      • Lind J.
      • Lymar S.V.
      • Merenyi G.
      • Radi R.
      Carbon dioxide-catalyzed peroxynitrite reactivity–The resilience of the radical mechanism after two decades of research.
      )) and to decompose to give NO2· and CO3-., and thereby generates radical-mediated products. As NO2· is formed from both ONOOH and ONOOCO2, nitrated products are commonly detected, with these being mainly generated from Tyr and Trp residues via dimerization reactions of NO2· with the TyrO and TrpN species formed by HO or CO3-. (
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). More limited modifications are detected from radical chemistry at Phe and His (
      • Ischiropoulos H.
      • al-Mehdi A.B.
      Peroxynitrite-mediated oxidative protein modifications.
      ,
      • Alvarez B.
      • Radi R.
      Peroxynitrite reactivity with amino acids and proteins.
      ), but oxidation of Cys and Met likely occurs via both one- and two-electron reactions (
      • Alvarez B.
      • Radi R.
      Peroxynitrite reactivity with amino acids and proteins.
      ).
      With hypohalous acids (HOCl, etc.), reaction occurs most rapidly with the sulfur amino acids (Cys > Met > cystine) to give a mixture of species (Table 1, Table 2) (
      • Hawkins C.L.
      • Pattison D.I.
      • Davies M.J.
      Hypochlorite-induced oxidation of amino acids, peptides and proteins.
      ). Reaction also occurs, albeit less rapidly, with nitrogen nucleophiles (i.e. His, the α-amino group, and Lys) to give short-lived chloramines (RNHCl) (
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      ,
      • Hawkins C.L.
      • Pattison D.I.
      • Davies M.J.
      Hypochlorite-induced oxidation of amino acids, peptides and proteins.
      ,
      • Thomas E.L.
      Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli.
      ) that retain the oxidizing capacity of HOCl but react much less rapidly and with greater selectivity (
      • Thomas E.L.
      Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli.
      ,
      • Peskin A.V.
      • Midwinter R.G.
      • Harwood D.T.
      • Winterbourn C.C.
      Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity.
      ). Reaction also occurs with Trp and Tyr, although with lower rate constants (
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      ). With Trp, oxygenated (and possibly chlorinated) species are formed (
      • Hawkins C.L.
      • Pattison D.I.
      • Davies M.J.
      Hypochlorite-induced oxidation of amino acids, peptides and proteins.
      ), and with Tyr, the major product is 3-chloro-Tyr (
      • Kettle A.J.
      Neutrophils convert tyrosyl residues in albumin to chlorotyrosine.
      ), a well-established biomarker of this oxidant, even though this species is formed slowly and in low yield (Table 2) (
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      ).
      1O2 reacts primarily with sulfur (Cys, Met, and cystine) and aromatic residues (Trp, Tyr, and His) (
      • Pattison D.I.
      • Rahmanto A.S.
      • Davies M.J.
      Photo-oxidation of proteins.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ), with reaction at the former species to give the dimer (cystine) and oxygenated products (Cys oxyacids, Met sulfoxide, and oxygenated disulfides) (
      • Pattison D.I.
      • Rahmanto A.S.
      • Davies M.J.
      Photo-oxidation of proteins.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ). With Trp, Tyr, and His, the initial products are endoperoxides formed by cycloaddition reactions, with these subsequently undergoing ring opening to give hydroperoxides, oxygenated products, and further cyclized materials (Table 2) (
      • Pattison D.I.
      • Rahmanto A.S.
      • Davies M.J.
      Photo-oxidation of proteins.
      ,
      • Davies M.J.
      Singlet oxygen-mediated damage to proteins and its consequences.
      ). As with the radical chemistry of Trp and His, these reactions can result in ring opening reactions and a similar (complex) mixture of species. The products from these amino acids therefore do not allow the initial oxidant to be easily identified.
      From the above discussion it is clear that different oxidants have very different chemical behaviors and reaction kinetics, and these differences can be magnified or decreased by a range of other factors that influence oxidant selectivity. This is discussed further in the following section.

      Factors affecting oxidant selectivity

      The extent of damage by a particular oxidant can be modulated by multiple factors, including the accessibility of the oxidant to the target residue (e.g. Trp residues are often buried within protein structures and have limited solvent accessibility), and also electrostatic interactions with residues on the protein surface (e.g. the presence of charge on oxidants such as O2-. and CO3-.). Neutral species may induce greater damage than charged species, and also at more remote locations, due to the greater propensity of such species to traverse membranes and hence diffuse away from their site of generation. The neutral species may also be better electrophiles and provide better leaving groups. This is exemplified by the greater reactivity of HOCl over OCl and ONOOH over ONOO (
      • Pattison D.I.
      • Davies M.J.
      Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds.
      ,
      • Ferrer-Sueta G.
      • Campolo N.
      • Trujillo M.
      • Bartesaghi S.
      • Carballal S.
      • Romero N.
      • Alvarez B.
      • Radi R.
      Biochemistry of peroxynitrite and protein tyrosine nitration.
      ). Ionization of side-chain residues on a protein increases their electron density, increasing their capacity to act as a nucleophile and be more readily oxidized; thus, the thiolate anion, RS, is more reactive than the parent RSH (and similarly for the related selenium-containing amino acid, selenocysteine, Sec), and the Tyr phenolate anion is more readily oxidized than the neutral phenol (
      • Ferrer-Sueta G.
      • Manta B.
      • Botti H.
      • Radi R.
      • Trujillo M.
      • Denicola A.
      Factors affecting protein thiol reactivity and specificity in peroxide reduction.
      ,
      • Criado S.
      • Soltermann A.T.
      • Marioli J.M.
      • García N.A.
      Sensitized photooxidation of di- and tripeptides of tyrosine.
      ).
      Readily oxidized residues (e.g. Cys, Trp, and Tyr) can undergo long-range electron transfer reactions and thereby function as radical “sinks” both within and between proteins (
      • Butler J.
      • Land E.J.
      • Prutz W.A.
      • Swallow A.J.
      Charge transfer between tryptophan and tyrosine in proteins.
      • Prütz W.A.
      • Butler J.
      • Land E.J.
      • Swallow A.J.
      The role of sulphur peptide functions in free radical transfer: a pulse radiolysis study.
      ,
      • Prütz W.A.
      • Land E.J.
      Charge transfer in peptides. Pulse radiolysis investigation of one-electron reactions in dipeptides of tryptophan and tyrosine.
      ,
      • Prüutz W.A.
      • Siebert F.
      • Butler J.
      • Land E.J.
      • Menez A.
      • Montenay-Garestier T.
      Charge transfer in peptides. Intramolecular radical transformations involving methionine, tryptophan and tyrosine.
      ). Such transfers can occur over long distances (e.g. in ribonucleotide reductase, DNA photolyase, and photosystem II), such that initial oxidation at one site can be rapidly transferred to another remote residue, with the electron transfer occurring through bonds or space (Fig. 5) (
      • Aubert C.
      • Vos M.H.
      • Mathis P.
      • Eker A.P.
      • Brettel K.
      Intraprotein radical transfer during photoactivation of DNA photolyase.
      ,
      • Barry B.A.
      • Chen J.
      • Keough J.
      • Jenson D.
      • Offenbacher A.
      • Pagba C.
      Proton coupled electron transfer and redox active tyrosines: structure and function of the tyrosyl radicals in ribonucleotide reductase and photosystem II.
      ). Consequently, the initial site of oxidation may not be the final site of modification. As the one-electron reduction potentials of Trp and Tyr are similar, radical formation at one residue can result in equilibration between residues, assuming suitable electron transfer pathways are available (
      • Prütz W.A.
      • Butler J.
      • Land E.J.
      • Swallow A.J.
      The role of sulphur peptide functions in free radical transfer: a pulse radiolysis study.
      ). One consequence of this is that radical termination reactions (e.g. dimerization of two TyrO, two TrpN, or cross-reaction of these species with NO2· to give nitrated products) may occur via the most accessible, or reactive, TyrO or TrpN rather than at the site of initial radical generation. Thus, cross-link formation involving Tyr and Trp radicals, and formation of products such as 3-nitro-Tyr, may be determined by the accessibility and reactivity of a particular residue, rather than the extent of initial reaction at that site (Fig. 5).
      Figure thumbnail gr5
      Figure 5Initial oxidation at electron-rich sites (e.g. Tyr and Trp residues but also Met, His, and Cys) can result in rapid electron transfer both within, and between, protein molecules. This can result in subsequent reactions and products being formed at sites that were not the initial site of oxidation and at locations remote from the initial site.
      In the light of these data, the next section summarizes commonly used methods to detect protein alterations, starting with modifications at the intact protein level (i.e. changes that markedly affect protein mass and structure: “gross changes”) and then progressing to techniques that identify and quantify changes at an amino acid level, and at specific sites within a protein sequence.

      Detection and quantification of protein oxidation

      Gross modification of parent proteins

      Oxidation of proteins can generate both fragmentation and aggregation of proteins. The latter can involve both covalent cross-linking as well as noncovalent interactions. Separation methods based on mass or charge (e.g. one- or two-dimensional electrophoresis and column chromatography) with subsequent detection methods (e.g. silver staining or immunoblotting) can provide limited information about such changes. This works best with purified proteins or limited mixtures, but it has severe limitations with complex samples and also when comparing healthy versus diseased samples, as the protein pools may be very different in such cases, even when two-dimensional gels are used to enhance resolution. Immunoblotting with specific antibodies can provide high sensitivity and specificity detection, but this approach is severely limited with regard to both the quantification and identification of modifications. Artifactual proteolysis or aggregation is also a serious concern. Both oxidant-mediated fragmentation and aggregation can be investigated using these approaches, but as fragmentation is often nonspecific or poorly-specific, discrete bands or spots (from 2D gels) are rare, with “band smears” being the usual outcome.
      Aggregation or cross-linking is more readily analyzed, as dimers (for example) generated by any pairing of residues are likely to provide bands/spots of similar mass. Care clearly needs to be taken as multiple proteins are typically present in each band or fraction. Reduced antibody recognition of a specific native epitope can be used as a method of assessing modification to that site, in either immunoblotting studies or ELISA. These approaches are limited by the availability of specific antibodies but have been used successfully in a large number of studies ranging from isolated proteins to tissues, and they have the advantage of very-high sensitivity. Increased information can be obtained if antibodies against both parent protein epitopes, and specific products (see below), are available (
      • Kato Y.
      • Wu X.
      • Naito M.
      • Nomura H.
      • Kitamoto N.
      • Osawa T.
      Immunochemical detection of protein dityrosine in atherosclerotic lesion of apo-E–deficient mice using a novel monoclonal antibody.
      • Al-Hilaly Y.K.
      • Biasetti L.
      • Blakeman B.J.
      • Pollack S.J.
      • Zibaee S.
      • Abdul-Sada A.
      • Thorpe J.R.
      • Xue W.-F.
      • Serpell L.C.
      • Goedert M.
      • Wright J.A.
      • Wang X.
      • Brown D.R.
      • Celej M.S.
      • Winner B.
      • et al.
      The involvement of dityrosine crosslinking in α-synuclein assembly and deposition in Lewy bodies in Parkinson’s disease.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Hammer A.
      • Malle E.
      • Davies M.J.
      Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Kawasaki H.
      • Hammer A.
      • Malle E.
      • Yamakura F.
      • Davies M.J.
      Peroxynitrite-mediated oxidation of plasma fibronectin.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Mariotti M.
      • Hammer A.
      • Hoefler G.
      • Hägglund P.
      • Malle E.
      • Wise S.G.
      • Davies M.J.
      Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function.
      ,
      • Trnka M.J.
      • Baker P.R.
      • Robinson P.J.
      • Burlingame A.L.
      • Chalkley R.J.
      Matching cross-linked peptide spectra: only as good as the worse identification.
      ,
      • Tiwari M.K.
      • Leinisch F.
      • Sahin C.
      • Møller I.M.
      • Otzen D.E.
      • Davies M.J.
      • Bjerrum M.J.
      Early events in copper-ion catalyzed oxidation of α-synuclein.
      ,
      • Fuentes-Lemus E.
      • Silva E.
      • Leinisch F.
      • Dorta E.
      • Lorentzen L.G.
      • Davies M.J.
      • López-Alarcon C.
      α- and β-casein aggregation induced by riboflavin-sensitized photo-oxidation occurs via di-tyrosine cross-links and is oxygen concentration dependent.
      ). An example of this approach are the studies that have examined HOCl-mediated damage to the extracellular matrix underlying endothelial cells. Binding of three specific antibodies (anti-fibronectin, anti-laminin, and anti-thrombospondin) was decreased on treatment with HOCl, implicating damage to these proteins (
      • Vissers M.C.
      • Thomas C.
      Hypochlorous acid disrupts the adhesive properties of subendothelial matrix.
      ). However, analysis of such data can be complex, as damage may also enhance antibody binding by exposing cryptic epitopes. Thus, low doses of HOCl appear to increase the affinity of anti-fibronectin antibodies to plasma fibronectin, whereas high concentrations have the opposite effect (
      • Olszowski S.
      • Olszowska E.
      • Kusior D.
      • Piwowarczyk M.
      • Stelmaszynska T.
      Hypochlorite action on plasma fibronectin promotes its extended conformation in complexes with antibodies.
      ). This has been rationalized in terms of the generation of an extended fibronectin conformation at low HOCl doses, and aggregation with high concentrations.
      An absence of positive data from ELISA or immunoblotting studies does not preclude the presence of damage, as epitopes may become inaccessible on protein oligomerization or as a result of other structural changes. Quantification is also challenging as this is very dependent on the sensitivity of the antibody: strong signals may be detected for low-abundance material, whereas abundant species may give a weak (or no) signal with a poor antibody. Separation of modified species by HPLC, for example, has been employed successfully with oxidants that are highly selective and that induce damage at a limited set of residues. An example is the separation of modified apolipoproteins AI and AII (
      • Pankhurst G.
      • Wang X.L.
      • Wilcken D.E.
      • Baernthaler G.
      • Panzenböck U.
      • Raftery M.
      • Stocker R.
      Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.
      ,
      • Garner B.
      • Witting P.K.
      • Waldeck A.R.
      • Christison J.K.
      • Raftery M.
      • Stocker R.
      Oxidation of high density lipoproteins. I. Formation of methionine sulfoxide in apolipoproteins AI and AII is an early event that accompanies lipid peroxidation and can be enhanced by α-tocopherol.
      ) after mild oxidation of high-density lipoproteins or plasma, with loss of the parent isoforms, and the formation of newly-oxidized species, as detected by HPLC (
      • Pankhurst G.
      • Wang X.L.
      • Wilcken D.E.
      • Baernthaler G.
      • Panzenböck U.
      • Raftery M.
      • Stocker R.
      Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein.
      ). Subsequent MS analysis of the fractions identified the modifications as loss of parent Met and generation of the corresponding sulfoxide.
      Other biophysical techniques (e.g. CD, light scattering, small-angle neutron scattering, small-angle X-ray scattering, turbidity methods, X-ray crystallography, and NMR spectroscopy) can also yield information on protein structure, particularly the generation of fragments or aggregates, as these methods are sensitive to changes in protein mass, the size of particles, modified secondary structure, and altered charge and solubility. X-ray crystallographic studies have provided evidence for increased electron density between residues in aggregated proteins supporting the presence and identification of particular cross-links and their nature (exact site of linkage and intra- versus inter-molecular; see, for example, data for oxidized peroxiredoxin 5, thioredoxin 2, and γS-crystallin (
      • Evrard C.
      • Capron A.
      • Marchand C.
      • Clippe A.
      • Wattiez R.
      • Soumillion P.
      • Knoops B.
      • Declercq J.P.
      Crystal structure of a dimeric oxidized form of human peroxiredoxin 5.
      ,
      • Smeets A.
      • Evrard C.
      • Landtmeters M.
      • Marchand C.
      • Knoops B.
      • Declercq J.P.
      Crystal structures of oxidized and reduced forms of human mitochondrial thioredoxin 2.
      ,
      • Thorn D.C.
      • Grosas A.B.
      • Mabbitt P.D.
      • Ray N.J.
      • Jackson C.J.
      • Carver J.A.
      The structure and stability of the disulfide-linked γS-crystallin dimer provide insight into oxidation products associated with lens cataract formation.
      )). However, with the exception of X-ray crystallography and NMR, these methods do not provide definitive information as to the sites and modifications, and these techniques are (currently) limited to homogeneous samples (often single proteins) with high modification levels.

      Total amino acid analysis

      This methodology can provide important quantitative data on the consumption of parent species, arising from all potential modification reactions, and for some species the yields of products can also be assessed (e.g. methionine sulfoxide, see below). Such data are important with regard to obtaining a material balance, something that has been difficult to achieve even with the simplest systems. An overview of this approach is provided in Fig. 6. Differences between loss and total product formation can provide vital information with regard to the generation of alternative (known or unknown) species (see also below). Typically, proteins are isolated (e.g. by precipitation from homogenates/lysates using TCA or organic solvents), cleaned up (e.g. delipidation), and subsequently subjected to hydrolysis to give the free amino acids and products (Fig. 6) (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). Processing is preferentially carried out in the presence of enzyme inhibitors and antioxidants to decrease artifacts (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). Proteolysis can be achieved using acid conditions (with this resulting in loss of Cys, cystine, and some Trp species, although this depends on the acid), alkaline conditions (which preserves Trp species, but results in loss of other species such as the product DOPA), or nonspecific proteases, such as Pronase (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Hammer A.
      • Malle E.
      • Davies M.J.
      Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.
      ,
      • Davies M.J.
      • Fu S.
      • Wang H.
      • Dean R.T.
      Stable markers of oxidant damage to proteins and their application in the study of human disease.
      ). The free amino acids (and any products) are then separated (e.g. by HPLC/UPLC) and quantified by mass spectrometry (MS), fluorescence (either directly, for some aromatic species, or by pre-column fluorescent tagging of free amino groups using reagents such as o-phthaldialdehyde, OPA), UV absorption, or electrochemical methods (Fig. 6) (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ,
      • Degendorfer G.
      • Chuang C.Y.
      • Hammer A.
      • Malle E.
      • Davies M.J.
      Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.
      ,
      • Davies M.J.
      • Fu S.
      • Wang H.
      • Dean R.T.
      Stable markers of oxidant damage to proteins and their application in the study of human disease.
      ). The combination of acid hydrolysis (using methane sulfonic acid) and OPA tagging allows data to be obtained for all common amino acids with the exception of Cys/cystine (which are acid-sensitive), Asn and Gln (which are converted to Asp and Glu, respectively), and Pro (which does not react with OPA, being an imine) (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). Enzymatic hydrolysis results in lower levels of artifactual oxidation due to the mild conditions (typically overnight incubation at 37 °C, pH 7.4) and hence preservation of acid/base-sensitive materials, but quantification can be problematic due to self-digestion of the protease resulting in release of additional amino acids although this is often limited (
      • Degendorfer G.
      • Chuang C.Y.
      • Hammer A.
      • Malle E.
      • Davies M.J.
      Peroxynitrous acid induces structural and functional modifications to basement membranes and its key component, laminin.
      ).
      Figure thumbnail gr6
      Figure 6Workflow to assess protein amino acid composition and their associated modifications. Proteins isolated and purified before digestion or hydrolysis to their constituent amino acids. Free amino acids and/or related oxidation products are then separated by LC. For some applications, pre-column or post-column derivatization of the amino acids and related products is required before separation to enable detection and quantification using one or more detection methods, which typically include MS, fluorescence, UV or visible absorption, or electrochemical (EC) detection. Abbreviations used are as follows: MSA, methane sulfonic acid; NFK, N-formylkynurenine.
      Quantitative data can be obtained by use of standard curves generated using amino acid mixtures, with heavy atom labeling (usually 2H, 13C, or 15N) in the case of MS analysis (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). Lys quantification can be problematic due to the second side-chain amino group, which results in multiple peaks if labeling (such as tagging with OPA or other amine-reactive tags) is incomplete. An internal standard (e.g. homo-Arg) allows sample recovery and derivatization efficiency to be assessed (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). The use of sacrificial oxidation targets (e.g. tryptamine), anoxic conditions, antioxidants, and other inhibitors are important to prevent significant losses, and data are typically normalized to nonmodified amino acids to compensate for any losses during processing (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). Comparison with expected amino acid compositions is recommended for studies using pure proteins, to ensure that materials are not lost (or the extent compensated for) during processing. Some modifications (e.g. Trp products during acid hydrolysis) are also known to be lost during processing (see below), which may result in an underestimation of the level of damage. MS analysis can also be readily used for studies on free amino acids, peptides, and for proteins. For the last of these, analysis is usually undertaken on loss of specific peptides after digestion using trypsin (or other enzymes) or at the intact protein level. Absolute quantification can, however, be tricky to achieve (see below).
      The total amino acid analysis approach is limited in that it only provides overall levels and not data on the sites of modifications within a sequence, nor the data on what proteins they might be present on, when analyzing complex mixtures. However, these data are an important complement to other approaches, such as MS peptide mass mapping, where only a limited number of species are typically analyzed.
      Loss of some amino acids can be quantified by alternative methods. Direct fluorescence (typically λex 280–285 nm and λem 340–345 nm) has been widely used to examine Trp residues (
      • Ehrenshaft M.
      • Deterding L.J.
      • Mason R.P.
      Tripping up Trp: modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.
      ), but this can be problematic, as Trp fluorescence is environment-sensitive (hence its use in examining protein unfolding (
      • Eftink M.R.
      The use of fluorescence methods to monitor unfolding transitions in proteins.
      )) and is difficult to use in the presence of other species that absorb or exhibit fluorescence at these wavelengths. This may include proteins with high concentrations of Tyr residues, heme proteins, species arising from Trp degradation, and some glycoxidation products. Lys and Arg can be quantified by reagents that give strongly fluorescent derivatives, such as fluorescamine (
      • Weigele M.
      • DeBarnardo S.L.
      • Tengi J.P.
      • Leimgruber W.
      A novel reagent for the fluorometric assay of primary amines.
      ) and 9,10-phenanthrenequinone (
      • Smith R.E.
      • MacQuarrie R.
      A sensitive fluorometric method for the determination of arginine using 9,10-phenanthrenequinone.
      ) respectively. These methods are rapid, sensitive, and give limited artifacts during sample preparation, but neither reagent is entirely specific, and hence needs to be used with care.
      A number of methods have been developed to allow quantification of the loss of the key redox-sensitive amino acid Cys on proteins. Detection and quantification of Cys products are covered below. Methods include spectrophotometric (e.g. using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), Ellman’s reagent (
      • Ellman G.L.
      Tissue sulfhydryl groups.
      ,
      • Hu M.L.
      Measurement of protein thiol groups and glutathione in plasma.
      ), or 4,4′-dithiodipyridine (
      • Hansen R.E.
      • Østergaard H.
      • Nørgaard P.
      • Winther J.R.
      Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4′-dithiodipyridine.
      )), fluorometric (e.g. using ThioGlo 1, (10-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-9-methoxy-3-oxo-3H-naphthol[2,1-b]pyran-2-carboxylic acid methyl ester), a naphthopyranone maleimide derivative, and related species (
      • Sharov V.S.
      • Dremina E.S.
      • Galeva N.A.
      • Williams T.D.
      • Schöneich C.
      Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC-electrospray-tandem MS: selective protein oxidation during biological aging.
      ), biotinylated reagents with various detection methods, MS (
      • Schöneich C.
      • Sharov V.S.
      Mass spectrometry of protein modifications by reactive oxygen and nitrogen species.
      ), and click chemistry (
      • Liu Y.
      • Hou W.
      • Sun H.
      • Cui C.
      • Zhang L.
      • Jiang Y.
      • Wu Y.
      • Wang Y.
      • Li J.
      • Sumerlin B.S.
      • Liu Q.
      • Tan W.
      Thiol-ene click chemistry: a biocompatible way for orthogonal bioconjugation of colloidal nanoparticles.
      ,
      • Chen G.
      • Feng H.
      • Xi W.
      • Xu J.
      • Pan S.
      • Qian Z.
      Thiol-ene click reaction-induced fluorescence enhancement by altering the radiative rate for assaying butyrylcholinesterase activity.
      ). These approaches can be used both directly on complex samples to give an overall readout or after protein separation using 1D or 2D gels (
      • Baty J.W.
      • Hampton M.B.
      • Winterbourn C.C.
      Detection of oxidant sensitive thiol proteins by fluorescence labeling and two-dimensional electrophoresis.
      ) and HPLC/UPLC (
      • Sharov V.S.
      • Dremina E.S.
      • Galeva N.A.
      • Williams T.D.
      • Schöneich C.
      Quantitative mapping of oxidation-sensitive cysteine residues in SERCA in vivo and in vitro by HPLC-electrospray-tandem MS: selective protein oxidation during biological aging.
      ,
      • Schöneich C.
      • Sharov V.S.
      Mass spectrometry of protein modifications by reactive oxygen and nitrogen species.
      ). With fluorescent or biotinylated thiol derivatization reagents, care should be taken to ensure specificity of the probe (
      • Hawkins C.L.
      • Morgan P.E.
      • Davies M.J.
      Quantification of protein modification by oxidants.
      ). The use of MS with isotope-coded tags is a powerful method for determining the sites and extent of Cys oxidation in complex biological mixtures (reviewed in Ref.
      • Turecek F.
      Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.
      ).
      The oxidation of the major low-molecular-mass cellular thiol, glutathione (GSH), is widely employed as an oxidative stress marker (e.g. Ref.
      • Yang C.S.
      • Chou S.T.
      • Liu L.
      • Tsai P.J.
      • Kuo J.S.
      Effect of ageing on human plasma glutathione concentrations as determined by high-performance liquid chromatography with fluorimetric detection.
      ), particularly when expressed relative to its major oxidation product, the disulfide GSSG, or the total of these species (e.g. Ref.
      • Moriarty-Craige S.E.
      • Jones D.P.
      Extracellular thiols and thiol/disulfide redox in metabolism.
      ). As GSSG formation can be reversed by GSH reductase (
      • Meister A.
      Glutathione metabolism and its selective modification.
      ,
      • Deponte M.
      Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.
      ), this reaction can be exploited to determine levels of GSH and the total of GSSG and GSH spectrophotometrically. GSSG levels can then be determined by difference in the values. The GSH concentration can be determined as described above, and also via the consumption of NADPH, the co-factor for GSH reductase (
      • Meister A.
      Glutathione metabolism and its selective modification.
      ,
      • Deponte M.
      Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes.
      ). GSSG levels can also be determined using genetically-encoded probes, such as roGFP fused to glutaredoxin; these probes are not responsive to thioredoxin-mediated changes probably for steric reasons. These probes allow real-time imaging of redox changes at specific and defined locations within living cells, although they also have some significant caveats; in particular, these are not direct oxidant probes, for they only report on the redox status of the specific environment being examined (
      • Albrecht S.C.
      • Sobotta M.C.
      • Bausewein D.
      • Aller I.
      • Hell R.
      • Dick T.P.
      • Meyer A.J.
      Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes.
      ,
      • Ezeriņa D.
      • Morgan B.
      • Dick T.P.
      Imaging dynamic redox processes with genetically encoded probes.
      ). Kits are available for such measurements. Direct measurements of both GSH and GSSG can be achieved after separation (e.g. by HPLC/UPLC) with various detection methods, including electrochemistry (e.g. Ref.
      • Richie Jr., J.P.
      • Lang C.A.
      The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection.
      ) and fluorescent tagging (e.g. with dansyl chloride or monobromobimane (
      • Martin J.
      • White I.N.
      Fluorimetric determination of oxidised and reduced glutathione in cells and tissues by high-performance liquid chromatography following derivatization with dansyl chloride.
      )). The levels and ratio of Cys/cystine and other thiols present in plasma have also been used as oxidative stress indicators in plasma (
      • Johnson J.M.
      • Strobel F.H.
      • Reed M.
      • Pohl J.
      • Jones D.P.
      A rapid LC-FTMS method for the analysis of cysteine, cystine and cysteine/cystine steady-state redox potential in human plasma.
      ). The absolute amounts, and ratio, of protein-bound Cys and low-molecular-mass thiols can be individually assessed after separation of the high- and low-molecular-weight fractions (e.g. by use of spin filters or protein precipitation).
      Reduced and oxidized thiols on proteins separated on 1D or 2D gels can be assessed by a number of methods (reviewed in Ref.
      • Eaton P.
      Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures.
      ), including the use of fluorescent-tagging (e.g. 5-iodoacetamidofluorescein, a fluorescent derivative of iodoacetamide) or biotin-tagging (e.g. Ref.
      • Baty J.W.
      • Hampton M.B.
      • Winterbourn C.C.
      Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells.
      ). Reversible thiol modifications can be examined by use of derivatization reagents after oxidant treatment, reduction of the reversible modification, and use of a second orthogonal tag (also see below and Ref.
      • Baty J.W.
      • Hampton M.B.
      • Winterbourn C.C.
      Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells.
      ).
      Direct quantification of disulfides (e.g. cystine) is complex but can be achieved by mild protein digestion methods (e.g. using chemicals such as cyanogen bromide or enzymatic approaches), which cleave the polypeptide backbone between the half-cystinyl residues, under conditions that minimize thiol-disulfide exchange and disulfide reduction. Diagonal electrophoresis was used in early studies (reviewed in Ref.
      • Creighton T.E.
      Disulfide bond formation in proteins.
      ), but this is now often examined using MS partial digestion and LC separation (e.g. Ref.
      • Yazdanparast R.
      • Andrews P.
      • Smith D.L.
      • Dixon J.E.
      A new approach for detection and assignment of disulfide bonds in peptides.
      ). Other MS methods have also been developed for cystine (
      • Johnson J.M.
      • Strobel F.H.
      • Reed M.
      • Pohl J.
      • Jones D.P.
      A rapid LC-FTMS method for the analysis of cysteine, cystine and cysteine/cystine steady-state redox potential in human plasma.
      ).

      Detection of protein oxidation intermediates

      Modification of proteins and peptides by oxidants can result in the generation of a number of intermediate species that have modest lifetimes and stabilities. As the assay of these materials requires specialized methods, these are discussed separately from the analysis of long-lived (“stable”) products, which are discussed later in this review. An overview of these methods is provided in Fig. 7.
      Figure thumbnail gr7
      Figure 7Approaches and experimental methods to detect reactive intermediates on proteins.

      Radicals

      Radicals, both initiating species and those formed on peptides and proteins, can be detected by a number of approaches including direct spectroscopic methods such as UV-visible, resonance Raman, conductivity, and electron paramagnetic resonance (EPR). Because of the short half-lives of most radicals, techniques with rapid response times are required, and of the above methods, only EPR is specific for radicals (
      ). Measurements using other techniques can be readily confounded by more abundant nonradical species, and hence they are only typically used with very clean systems coupled to rapid radical generation methods (e.g. pulse radiolysis, flash photolysis, and stopped flow) (
      ). These other methods, although limited in applicability, can provide valuable kinetic (rate constant) data.
      The use of EPR spectroscopy to detect and identify amino acid, peptide, and protein radicals in both isolated and complex systems has been reviewed elsewhere (
      • Davies M.J.
      • Hawkins C.L.
      EPR spin trapping of protein radicals.
      ,
      • Hawkins C.L.
      • Davies M.J.
      Detection and characterisation of radicals in biological materials using EPR methodology.
      ,
      • Davies M.J.
      Detection and characterisation of radicals using electron paramagnetic resonance (EPR) spin trapping and related methods.
      ). Although this is a very powerful analytical technique for identification of radicals (the “gold standard”), quantification is very challenging due to the short lifetime of these species. This can be (partially) overcome by use of ancillary techniques such as rapid-flow methods, in situ photolysis or radiolysis, freeze-quenching, and spin trapping (e.g. using nitrone compounds, such as 5,5-dimethyl-1-pyrroline N-oxide, DMPO), although each of these has advantages and disadvantages (reviewed in Refs.
      • Davies M.J.
      • Hawkins C.L.
      EPR spin trapping of protein radicals.
      ,
      • Hawkins C.L.
      • Davies M.J.
      Detection and characterisation of radicals in biological materials using EPR methodology.
      ,
      • Davies M.J.
      Detection and characterisation of radicals using electron paramagnetic resonance (EPR) spin trapping and related methods.
      ). Spin trapping is the most widely used as it allows studies on fluids, cells, tissues, and intact animals (e.g. mice and rats; see e.g. Refs.
      ,
      • Hawkins C.L.
      • Davies M.J.
      Detection and characterisation of radicals in biological materials using EPR methodology.
      ,
      • Davies M.J.
      Detection and characterisation of radicals using electron paramagnetic resonance (EPR) spin trapping and related methods.
      ). In this technique, a compound (the spin trap, typically a nitroso or nitrone) is added, with the aim of generating more stable, detectable adducts (Fig. 7). Analysis of the resulting spectra can then yield information on the species present (
      • Davies M.J.
      • Hawkins C.L.
      EPR spin trapping of protein radicals.
      ,
      • Hawkins C.L.
      • Davies M.J.
      Detection and characterisation of radicals in biological materials using EPR methodology.
      ,
      • Davies M.J.
      Detection and characterisation of radicals using electron paramagnetic resonance (EPR) spin trapping and related methods.
      ). The technique is artifact-prone and needs to be carried out with care. The resulting data can be definitive with regard to the species present, but also have a number of inherent caveats. In particular, the sensitivity of the method allows minor pathways to be detected and potentially misinterpreted, and the data do not provide information as to the absolute radical concentrations. Differences in the rates of trapping (or adduct decay) may make a minor species appear important when compared with a major pathway that yields very transient species. The long lifetimes of some protein–radical adducts have allowed this approach to be combined with other analytical methods, including MS, to provide detailed information (reviewed in Ref.
      • Davies M.J.
      • Hawkins C.L.
      EPR spin trapping of protein radicals.
      ).
      A related method, immunospin trapping, has been developed to detect protein radicals (reviewed in Refs.
      • Mason R.P.
      • Ganini D.
      Immuno-spin trapping of macromolecules free radicals in vitro and in vivo–One stop shopping for free radical detection.
      ,
      • Mason R.P.
      Imaging free radicals in organelles, cells, tissue, and in vivo with immuno-spin trapping.
      ), with this method utilizing the decay of a nitroxide spin adduct to a nitrone species to generate an antigen that is recognized by an antibody. This then allows the sensitive and specific detection of the former radical species, on isolated proteins, in cells, and in animals (primarily rodents). Immunospin trapping system can also be combined with LC/MS/MS and with the anti-DMPO antibody used to screen separate fractions (e.g. from HPLC or size-exclusion columns) for the presence of adducted DMPO. This allows residues that previously contained a radical to be readily detected, as the mass addition arising from the presence of DMPO can be readily detected (
      • Mason R.P.
      • Ganini D.
      Immuno-spin trapping of macromolecules free radicals in vitro and in vivo–One stop shopping for free radical detection.
      ,
      • Mason R.P.
      Imaging free radicals in organelles, cells, tissue, and in vivo with immuno-spin trapping.
      ).

      Hydroperoxides

      Hydroperoxides are major products of both radical- and 1O2-mediated damage to proteins in the presence of O2. These species are formed on many side chains in high yield by a wide range of insults, including oxygen-derived radicals, 1O2, activated white cells, ONOOH, and metal ion-catalyzed systems (Table 2) (reviewed in Ref.
      • Davies M.J.
      Protein oxidation and peroxidation.
      ). Decomposition of these species, which have lifetimes of minutes to many hours, by metal ions or UV light can give further radicals (RO, R, and ROO) that propagate damage, including to lipids, other proteins, DNA, and RNA (
      • Davies M.J.
      Protein oxidation and peroxidation.
      ,
      • Luxford C.
      • Morin B.
      • Dean R.T.
      • Davies M.J.
      Histone H1- and other protein- and amino acid-hydroperoxides can give rise to free radicals which oxidize DNA.
      ,
      • Luxford C.
      • Dean R.T.
      • Davies M.J.
      Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA.
      ). Two-electron reduction by both low-molecular-mass reductants (e.g. GSH) and some protein and enzyme systems (
      • Morgan P.E.
      • Dean R.T.
      • Davies M.J.
      Protective mechanisms against peptide and protein peroxides generated by singlet oxygen.
      ) gives the corresponding (stable) alcohols as products. In contrast, reaction of ROOH/H2O2 with critical Cys residues present on proteins or enzymes can inhibit enzyme activity and exacerbate damage (
      • Devarie-Baez N.O.
      • Silva Lopez E.I.
      • Furdui C.M.
      Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ,
      • Morgan P.E.
      • Dean R.T.
      • Davies M.J.
      Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.
      ,
      • Dremina E.S.
      • Sharov V.S.
      • Davies M.J.
      • Schöneich C.
      Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid peroxides.
      ). Alcohols consistent with the formation and subsequent decay of hydroperoxides have been detected in healthy and disease specimens (e.g. human lens cataracts (
      • Fu S.
      • Dean R.
      • Southan M.
      • Truscott R.
      The hydroxyl radical in lens nuclear cataractogenesis.
      ) and atherosclerotic lesions (
      • Fu S.
      • Davies M.J.
      • Stocker R.
      • Dean R.T.
      Evidence for roles of radicals in protein oxidation in advanced human atherosclerotic plaque.
      )), consistent with the occurrence of this chemistry in vivo. Hydroperoxides can be quantified by multiple methods, including iodometric titration, the ferrous oxidation–xylenol orange (FOX) assay, and also by use of boronic acid probes (Fig. 7) (
      • Jessup W.
      • Dean R.T.
      • Gebicki J.M.
      Iodometric determination of hydroperoxides in lipids and proteins.
      ,
      • Gay C.A.
      • Gebicki J.M.
      Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method.
      ,
      • Wolff S.P.
      Ferrous ion oxidation in the presence of ferric ion indicator xylenol orange for measurement of hydroperoxides.
      ). In the first of these, reaction of ROOH with iodide ions (I) in the presence of acid generates triiodide (I3) that can be quantified by its absorbance at 358 nm. This method is quantitative and has a well-defined 1:1 stoichiometry, but it needs be performed under strict anoxic conditions due to the sensitivity of acidified iodide solutions to O2; it is therefore technically demanding (
      • Jessup W.
      • Dean R.T.
      • Gebicki J.M.
      Iodometric determination of hydroperoxides in lipids and proteins.
      ). The FOX (ferrous oxidation–xylenol orange) method assays hydroperoxide-mediated oxidation of a Fe(II)–xylenol orange complex to the Fe(III) form, with the latter quantified via its absorbance at 560 nm (
      • Gay C.A.
      • Gebicki J.M.
      Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method.
      ,
      • Wolff S.P.
      Ferrous ion oxidation in the presence of ferric ion indicator xylenol orange for measurement of hydroperoxides.
      ). This assay is generic for all hydroperoxides, and also H2O2 (so samples are typically pre-treated with catalase to remove this species), and has been adapted to allow quantification of both protein- and lipid-derived hydroperoxides (
      • Gay C.A.
      • Gebicki J.M.
      Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method.
      ). This method has a low sensitivity to O2, but it is not compatible with some buffers and has a poorly-defined stoichiometry; consequently, data are typically reported as H2O2 equivalents obtained by use of a standard curve generated using this species (
      • Bou R.
      • Codony R.
      • Tres A.
      • Decker E.A.
      • Guardiola F.
      Determination of hydroperoxides in foods and biological samples by the ferrous oxidation-xylenol orange method: a review of the factors that influence the method's performance.
      ). Boronic acid probes that give fluorescent products on reaction with hydroperoxides have also been introduced (
      • Michalski R.
      • Zielonka J.
      • Gapys E.
      • Marcinek A.
      • Joseph J.
      • Kalyanaraman B.
      Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.
      ), and these can provide real-time data, although they may be limited to in vitro systems, as multiple other oxidants also react with these pro-fluorescent species (
      • Hardy M.
      • Zielonka J.
      • Karoui H.
      • Sikora A.
      • Michalski R.
      • Podsiadły R.
      • Lopez M.
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Ouari O.
      Detection and characterization of reactive oxygen and nitrogen species in biological systems by monitoring species-specific products.
      ,
      • Zielonka J.
      • Sikora A.
      • Hardy M.
      • Joseph J.
      • Dranka B.P.
      • Kalyanaraman B.
      Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides.
      ).

      Chloramines/bromamines

      Reaction of nucleophilic nitrogen centers (e.g. imidazole, amines, and amides), with hypohalous acids (HOCl and HOBr) generates N-chloro and N-bromo species (RNHX, where X = Cl, Br (
      • Davies M.J.
      • Hawkins C.L.
      • Pattison D.I.
      • Rees M.D.
      Mammalian heme peroxidases: from molecular mechanisms to health implications.
      ,
      • Weiss S.J.
      • Lampert M.B.
      • Test S.T.
      Long-lived oxidants generated by human neutrophils: characterization and bioactivity.
      )). These species can be formed on both the N-terminal amine and the side chains of Lys and His and to lesser extent Arg, Asn, Gln, and backbone amides (
      • Davies M.J.
      • Hawkins C.L.
      • Pattison D.I.
      • Rees M.D.
      Mammalian heme peroxidases: from molecular mechanisms to health implications.
      ). Quantification is possible by their UV absorption bands (Fig. 7) (λ ~250 and ~290 nm, for RNHCl and RNHBr respectively (
      • Thomas E.L.
      • Grisham M.B.
      • Jefferson M.M.
      Preparation and characterization of chloramines.
      )), but these overlap with many other species in complex systems, so quantification is usually achieved by reaction with an added probe such as 5-thio-2-nitrobenzoic acid (TNB, which is oxidized to the corresponding dimer, DTNB (
      • Thomas E.L.
      • Grisham M.B.
      • Jefferson M.M.
      Preparation and characterization of chloramines.
      )), with quantification achieved via the loss of absorbance from TNB at 412 nm. Oxidation of TNB to DTNB also occurs with many oxidants (e.g. HOCl, HOBr, HOSCN, H2O2, and other peroxides, ONOOH, 1O2, and many radicals), and hence it is not specific for any particular species. Iodometric titration can also be employed (see section on “Hydroperoxides”), but again this lacks specificity. Iodide ions have also been used to catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine and dihydrorhodamine, by chloramines, to optically absorbing or fluorescent products (Fig. 7) (
      • Dypbukt J.M.
      • Bishop C.
      • Brooks W.M.
      • Thong B.
      • Eriksson H.
      • Kettle A.J.
      A sensitive and selective assay for chloramine production by myeloperoxidase.
      ); this method is more specific but also has some drawbacks, including slow reaction (
      • Dypbukt J.M.
      • Bishop C.
      • Brooks W.M.
      • Thong B.
      • Eriksson H.
      • Kettle A.J.
      A sensitive and selective assay for chloramine production by myeloperoxidase.
      ). N-Chloro species have also been identified by LC/MS, but the instability of these species at elevated temperatures limits quantitative assessment (
      • Raftery M.J.
      Detection and characterization of N-α-chloramines by electrospray tandem mass spectrometry.
      ).

      Sulfenic acids and related species

      Sulfenic acids (RSOH, with the formation of these species often termed S-sulfenylation), sulfenyl chlorides (RSCl), and S-nitrosated species (RSNO, S-nitrosylated) are major intermediates formed by two-electron oxidants with Cys residues and related species (
      • Poole L.B.
      The basics of thiols and cysteines in redox biology and chemistry.
      ,
      • Devarie-Baez N.O.
      • Silva Lopez E.I.
      • Furdui C.M.
      Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ,
      • Yang J.
      • Gupta V.
      • Tallman K.A.
      • Porter N.A.
      • Carroll K.S.
      • Liebler D.C.
      Global, in situ, site-specific analysis of protein S-sulfenylation.
      ). S-Nitrosylated and S-nitrated (RSNO2) species are also formed by one-electron mechanisms involving reaction of RS with NO and NO2·, respectively. RSOH and the corresponding sulfenyl amides RS-NHC(O)R′ (
      • Takakura K.
      • Beckman J.S.
      • MacMillan-Crow L.A.
      • Crow J.P.
      Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1b, CD45, and LAR by peroxynitrite.
      ) are key intermediates in the catalytic and regulatory processes of some proteins and enzymes (
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ), particularly as these are formed in a reversible manner and hence may provide protection against irreversible oxidation of critical Cys residues and provide a facile “on-off” switch for enzyme activity and act as redox switches (
      • Ross S.H.
      • Lindsay Y.
      • Safrany S.T.
      • Lorenzo O.
      • Villa F.
      • Toth R.
      • Clague M.J.
      • Downes C.P.
      • Leslie N.R.
      Differential redox regulation within the PTP superfamily.
      ). Most of these species (with the exception of RSNO) react rapidly with other thiols (and other nucleophiles and oxidants) to give disulfides, thiosulfinates, and sulfinic (RSO2H) or sulfonic acids (RSO3H) (
      • Hogg D.R.
      The Chemistry of Sulphenic Acids and Their Derivatives.
      ). The higher oxyacids are usually irreversible oxidation products, although RSO2H can be reduced by sulfiredoxins (
      • Akter S.
      • Fu L.
      • Jung Y.
      • Conte M.L.
      • Lawson J.R.
      • Lowther W.T.
      • Sun R.
      • Liu K.
      • Yang J.
      • Carroll K.S.
      Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.
      ). As sulfenic acids are key redox switches, considerable effort has been expended in developing methods to quantify these intermediates (
      • Akter S.
      • Fu L.
      • Jung Y.
      • Conte M.L.
      • Lawson J.R.
      • Lowther W.T.
      • Sun R.
      • Liu K.
      • Yang J.
      • Carroll K.S.
      Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.
      ,
      • Li R.
      • Kast J.
      Biotin switch assays for quantitation of reversible cysteine oxidation.
      ). Most methods for the detection of RSOH rely on chemical derivatization or trapping methods, with the prototypic species being 5,5-dimethyl-1,3-cyclohexanedione (dimedone), which reacts with RSOH (and other species (
      • Forman H.J.
      • Davies M.J.
      • Krämer A.C.
      • Miotto G.
      • Zaccarin M.
      • Zhang H.
      • Ursini F.
      Protein cysteine oxidation in redox signaling: caveats on sulfenic acid detection and quantification.
      )) to form a stable thioether adduct (
      • Poole L.B.
      The basics of thiols and cysteines in redox biology and chemistry.
      ,
      • Devarie-Baez N.O.
      • Silva Lopez E.I.
      • Furdui C.M.
      Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.
      ,
      • Yang J.
      • Carroll K.S.
      • Liebler D.C.
      The expanding landscape of the thiol redox proteome.
      ), which can be quantified by MS or by use of fluorescent or biotinylated tags (Fig. 7) (
      • Yang J.
      • Gupta V.
      • Tallman K.A.
      • Porter N.A.
      • Carroll K.S.
      • Liebler D.C.
      Global, in situ, site-specific analysis of protein S-sulfenylation.
      ,
      • Furdui C.M.
      • Poole L.B.
      Chemical approaches to detect and analyze protein sulfenic acids.
      ,
      • Gupta V.
      • Paritala H.
      • Carroll K.S.
      Reactivity, selectivity, and stability in sulfenic acid detection: a comparative study of nucleophilic and electrophilic probes.
      ,
      • Lo Conte M.
      • Lin J.
      • Wilson M.A.
      • Carroll K.S.
      A chemical approach for the detection of protein sulfinylation.
      ). Reduction of sulfenic acids by arsenite has been utilized to develop a “biotin-switch” method for labeling protein RSOH, with the reduced amino acid subsequently labeled with biotin-maleimide. The adducts can then be detected using immunoblotting with streptavidin-horseradish peroxidase or separated using streptavidin-agarose (
      • Saurin A.T.
      • Neubert H.
      • Brennan J.P.
      • Eaton P.
      Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.
      ).
      S-Nitrosated, sometimes (incorrectly) named as S-nitrosylated, Cys residues are also key signaling species and may act as a reservoir of NO (reviewed in Refs.
      • Janssen-Heininger Y.M.
      • Mossman B.T.
      • Heintz N.H.
      • Forman H.J.
      • Kalyanaraman B.
      • Finkel T.
      • Stamler J.S.
      • Rhee S.G.
      • van der Vliet A.
      Redox-based regulation of signal transduction: principles, pitfalls, and promises.
      ,
      • Chiesa J.J.
      • Baidanoff F.M.
      • Golombek D.A.
      Don't just say no: differential pathways and pharmacological responses to diverse nitric oxide donors.
      ). Protein S-nitrosation has also been implicated in multiple disease states, particularly those involving neurodegeneration and inflammation (reviewed in Refs.
      • Nakamura T.
      • Lipton S.A.
      Emerging roles of S-nitrosylation in protein misfolding and neurodegenerative diseases.
      ,
      • Jaffrey S.R.
      • Erdjument-Bromage H.
      • Ferris C.D.
      • Tempst P.
      • Snyder S.H.
      Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.
      ). The classical “biotin-switch” technique that has been widely used quantifies these species (
      • Jaffrey S.R.
      • Erdjument-Bromage H.
      • Ferris C.D.
      • Tempst P.
      • Snyder S.H.
      Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.
      ,
      • Devarie-Baez N.O.
      • Zhang D.
      • Li S.
      • Whorton A.R.
      • Xian M.
      Direct methods for detection of protein S-nitrosylation.
      ,
      • Chiappetta G.
      • Ndiaye S.
      • Igbaria A.
      • Kumar C.
      • Vinh J.
      • Toledano M.B.
      Proteome screens for Cys residues oxidation: the redoxome.
      ). In its classical form, nonmodified thiols are first blocked, and following the removal of excess alkylating reagent, ascorbate is added to selectively reduce any S-nitrosothiols (but not other species) to free thiols that are then labeled and detected by immunoblotting or fluorescent tagging following separation. Multiple iterations and improvements of this method have been proposed to enhance its specificity and to allow more rigorous quantification of RSNO levels. Many drawbacks have been reported, and considerable care and appropriate controls need to be employed to minimize artifacts (
      • Gladwin M.T.
      • Wang X.
      • Hogg N.
      Methodological vexation about thiol oxidation versus S-nitrosation.
      ,
      • Forrester M.T.
      • Foster M.W.
      • Stamler J.S.
      Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress.
      ).
      Multiple studies have reported MS methods to detect RSNO species (
      • Beuve A.
      • Wu C.
      • Cui C.
      • Liu T.
      • Jain M.R.
      • Huang C.
      • Yan L.
      • Kholodovych V.
      • Li H.
      Identification of novel S-nitrosation sites in soluble guanylyl cyclase, the nitric oxide receptor.
      ,
      • Wu C.
      • Liu T.
      • Wang Y.
      • Yan L.
      • Cui C.
      • Beuve A.
      • Li H.
      Biotin switch processing and mass spectrometry analysis of S-nitrosated thioredoxin and its transnitrosation targets.
      ). As with other unstable intermediates, considerable care needs to be taken to avoid artifactual changes in the levels and sites of modification, as it is well-established that some RSNO species undergo ready transnitrosation reactions (
      • Wu C.
      • Liu T.
      • Wang Y.
      • Yan L.
      • Cui C.
      • Beuve A.
      • Li H.
      Biotin switch processing and mass spectrometry analysis of S-nitrosated thioredoxin and its transnitrosation targets.
      ,
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ,
      • Wynia-Smith S.L.
      • Smith B.C.
      Nitrosothiol formation and S-nitrosation signaling through nitric oxide synthases.
      ).
      In biological systems, nitrosation is readily reversed, and this appears to be primarily driven by enzymatic reactions with reduction of low-molecular-mass species, such as S-nitrosated GSH (GSNO), being catalyzed by the widespread enzyme S-nitrosoglutathione reductase (GSNOR) (
      • Lancaster Jr., J.R.
      How are nitrosothiols formed de novo in vivo?.
      ,
      • Wynia-Smith S.L.
      • Smith B.C.
      Nitrosothiol formation and S-nitrosation signaling through nitric oxide synthases.
      ,
      • Liu L.
      • Hausladen A.
      • Zeng M.
      • Que L.
      • Heitman J.
      • Stamler J.S.
      A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans.
      ), whereas removal of protein species is catalyzed by members of the thioredoxin family (
      • Benhar M.
      • Forrester M.T.
      • Hess D.T.
      • Stamler J.S.
      Regulated protein denitrosylation by cytosolic and mitochondrial thioredoxins.
      ).
      Persulfidation (RSSH formation) is a relatively recently identified modification of proteins that can act as both a redox control mechanism and sensor of redox stress (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ). These species are formed, at least in part, via downstream reactions of H2S, a relatively recent addition to the family of a gaseous signal transmitter family, the other members being NO and CO. Conversion of Cys to Cys-SSH occurs through sulfuration or persulfidation processes and may involve oxidized H2S species and particularly polysulfides (H2Sn), as well as other pathways. H2S is generated from Cys and homocysteine by widely-expressed enzymes, including cystathionine β-synthase, cystathionine γ-lyase, cysteine aminotransferase, and 3-mercaptopyruvate sulfurtransferase. As these species are expressed in the vascular wall, H2S has been proposed as a regulator of vascular tone, neuronal health, the integrity of endothelial cells barriers, smooth muscle cell proliferation and survival, angiogenesis, and as a modulator of inflammation (
      • Filipovic M.R.
      • Zivanovic J.
      • Alvarez B.
      • Banerjee R.
      Chemical biology of H2S signaling through persulfidation.
      ,
      • Bełtowski J.
      Synthesis, metabolism, and signaling mechanisms of hydrogen sulfide: an overview.
      ,
      • Paul B.D.
      • Snyder S.H.
      Gasotransmitter hydrogen sulfide signaling in neuronal health and disease.
      ) Persulfides can be detected and quantified using a tag-switch method (
      • Zhang D.
      • Macinkovic I.
      • Devarie-Baez N.O.
      • Pan J.
      • Park C.M.
      • Carroll K.S.
      • Filipovic M.R.
      • Xian M.
      Detection of protein S-sulfhydration by a tag-switch technique.
      ); this should not be confused with the biotin-switch method (
      • Mustafa A.K.
      • Gadalla M.M.
      • Sen N.
      • Kim S.
      • Mu W.
      • Gazi S.K.
      • Barrow R.K.
      • Yang G.
      • Wang R.
      • Snyder S.H.
      H2S signals through protein S-sulfhydration.
      ), by MS methods, and also by use of fluorescent dyes, although the last of these, like most fluorescent dye approaches (
      • Hardy M.
      • Zielonka J.
      • Karoui H.
      • Sikora A.
      • Michalski R.
      • Podsiadły R.
      • Lopez M.
      • Vasquez-Vivar J.
      • Kalyanaraman B.
      • Ouari O.
      Detection and characterization of reactive oxygen and nitrogen species in biological systems by monitoring species-specific products.