High-throughput screening of cellular redox sensors using modern redox proteomics approaches.

Expert Review of Proteomics Downloaded from informahealthcare.com by Nyu Medical Center on 07/21/15 For personal use only.

Review

High-throughput screening of cellular redox sensors using modern redox proteomics approaches Expert Rev. Proteomics Early online, 1–13 (2015)

Jingwen Jiang‡1,2, Kui Wang‡1, Edouard C Nice3, Tao Zhang4 and Canhua Huang*1,2 1 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, PR China 2 Hainan Medical University, Haikou, 571199, PR China 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia 4 School of Biomedical Sciences, Chengdu Medical College, Chengdu 610500, PR China *Author for correspondence: Tel.: +86 132 583 703 46 Fax: +86 028 855 027 96 [email protected] ‡ These authors contributed equally to this work

informahealthcare.com

Cancer cells are characterized by higher levels of intracellular reactive oxygen species (ROS) due to metabolic aberrations. ROS are widely accepted as second messengers triggering pivotal signaling pathways involved in the process of cell metabolism, cell cycle, apoptosis, and autophagy. However, the underlying cellular mechanisms remain largely unknown. Recently, accumulating evidence has demonstrated that ROS initiate redox signaling through direct oxidative modification of the cysteines of key redox-sensitive proteins (termed redox sensors). Uncovering the functional changes underlying redox regulation of redox sensors is urgently required, and the role of different redox sensors in distinct disease states still remains to be identified. To assist this, redox proteomics has been developed for the high-throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for cancer treatment. Highlighted here are recent advances in redox proteomics approaches and their applications in identifying redox sensors involved in tumor development. KEYWORDS: Biotin switch . mass spectrometry . redox proteomics . reactive oxygen species . Thiol-trapping

Metabolic aberrations in cancer cells lead to the accumulation of reactive oxygen species (ROS) due to the imbalance of ROS generation and elimination [1]. ROS are characterized as highly reactive molecules that exist in three forms, namely hydroxyl free radicals, superoxide and the non-radical oxidant hydrogen peroxide (H2O2) [2]. These reactive species are byproducts of metabolic reactions in mitochondria, endoplasmic reticulum (ER) and peroxisomes under both physiological and pathological conditions [3]. Over the past decades, ROS were recognized as tumor inducers as evidenced by the fact that elevated levels of ROS promoted tumor cell proliferation [4]. However, increasing evidence suggests that the roles ROS play in cancer cells remains controversial. Different levels of ROS exert diverse effects in cell proliferation, apoptosis, metastasis and autophagy [5–8]. Specifically, low levels of ROS contribute to tumorigenesis either by stimulating DNA mutation or acting as second messengers involved in cancer cell survival [9,10], whereas high levels of ROS are linked to

10.1586/14789450.2015.1069189

damage of cellular components, leading to cancer cell death [9]. In addition, it is experimentally confirmed that ROS are also involved in other diseases, including virus infection, neurodegenerative diseases, cardiovascular diseases, and immune disorder [11–14]. Unraveling the molecular mechanisms underlying redox regulation in cancer cells is therefore important and will assist in the development of novel therapeutic strategies. Recent studies suggest that ROS initiate redox signaling via direct or indirect oxidative modification of redox sensors, which mainly occurs on sulfur-containing cysteine residues [15]. A wide range of proteins, including kinases (pyruvate kinase M2, AMPK, Akt, JNK1, p38), transcription factors (NF-kB, p53, FOXOs, Nrf-2, HIF-1a) and chaperones (Hsp33, Hsp70, ASNA1) [16–21] are characterized as redox sensors, the biological functions of which are determined by the different redox status of active cysteines [22]. The free thiols (Pr-SH) in cysteines susceptible to ROS can undergo diverse patterns of oxidative

2015 Informa UK Ltd

ISSN 1478-9450

1

Review

Jiang, Wang, Nice, Zhang & Huang

GSH

GSH Grx

NADPH

SH

SOH Arsenite

Trx S

DTT

GSH S S S


Srx

Oxidants

RNS SNO Ascorbate O S-S-R

SO2H

SSG

SO3H SH

O

S-S-R

due to the challenges of sample purification, thiol-labeling, site-specific identification and quantification of the dynamic modifications [33]. In this review, we will outline the feasible thiol-trapping and thiol enrichment approaches and highlight the recent redox proteomics strategies for the identification of potential redox sensors. Thiol-trapping strategies

Trapping the redox status of thiols is a critical procedure prior to screening redox sensors using redox proteomics. As Figure 1. Patterns of redox modification. Free thiols can be oxidized to sulfenylation several cysteine oxoforms are labile, artifi(Cys-SOH), disufide bonds (PrS-SrP), S-glutathionylation (PrS-SG), and SNO. These oxidacial oxidation could occur when proteins tive modifications are reversible and can be reduced by physiological reductants such as Grx, Trx, GSH, NADPH and Srx. are exposed to air. In addition, intracelGrx: Glutaredoxin; GSH: Glutathione; DTT: Dithiothreitol; NAPDH: Reduced nicotinamide lular antioxidant enzymes could inhibit adenine dinucleotide phosphate; SNO: S-nitrosothiol; Srx: Sulfiredoxin; RNS: Reactive the oxidative modifications [34]. Hence, nitrogen species; Trx: Thioredoxin. during the preparation of protein extracts, it is crucial to rapidly quench modifications, including sulfenylation (Pr-SOH) (the initial the free thiols to avoid artificial oxidation and maintain the products which are considered as metastable intermediates pro- bona fide intracellular redox status of the proteins [33]. duced prior to disulfide bond formation [PrS-SPr]), sulfinylation (Pr-SO2H), sulfonylation (Pr-SO3H), as well as Quenching free thiols by acidification intramolecular, intermolecular or mixed disulfide bonds Free thiols with low pKa can exist as thiolate anion (S ) at (PrS-SPr) and S-glutathionylation (PrS-SG) (FIGURE 1) [23]. Pr- neutral pH, a nucleophile (pKa = 4.7–5.4) susceptible to oxiSO2H, a product of excessive oxidative sulfinylation, is com- dants. Acidification can block the oxidation of thiolate anion, monly considered as an irreversible oxidative modification. thereby quenching the free thiols [35]. Acids such as perchloric However, more recently, it has been shown that the hyperoxi- acid, trichloroacetic acid (TCA), and sulfosalicylic acid can be dative sulfinylation of specific proteins, such as 2-Cys-contain- used to block the thiolate anion and denature the proteins. ing peroxiredoxin (Prx) isoforms, can be reversed via the ATP- Protonation can be extremely rapid, with a rate constant in the dependent reaction catalyzed by sulfiredoxin (Srx) [24,25]. As an range of 109M 1 s 1. Additionally, the denaturation of proalternative to ROS per se, the oxidation of proteins can be teins enables protons to access all free thiols. However, it has achieved through interaction with oxidized thioredoxin (Trx) been observed that the perchloric acid- and sulfosalicylic acidor Prx [17]. It is worth mentioning that Pr-SOH, PrS-SPr, and treated free thiols can be oxidized artificially [36], whereas TCAPrS-SG can be reversed by cellular reductive enzymes, such as treated free thiols show limited oxidation. TCA may therefore the glutaredoxin (Grx)or thioredoxin (Trx) systems [26–28]. In be the most suitable reagent for quenching free thiols. 10–20% addition to the above-mentioned cysteine oxoforms, S-nitrosy- (w/v) TCA is recommended for efficiently precipitating prolation (PrS-NO), another oxidative modification of cysteine teins. In addition, by combination with sodium deoxycholate caused by nitric oxide (NO)-derived reactive nitrogen species (0.02%, w/v), which has been reported as a co-precipitation (RNS), can also regulate the activity and stability of pro- reagent, TCA can precipitate low-abundance proteins [37]. Proteins [29]. S-nitrosylation is considered as one of the major tein extracts are most commonly acidified using TCA prior to mechanisms of NO function. Accumulated NO or sustained subsequent analysis of the redox status. RNS production contributes to several pathological processes, including tumorigenesis and neurodegeneration and cardiovas- Labeling different thiol oxoforms cular disorder [30–32]. Identification of the distinct redox modifi- The thiolate anion can react readily with alkylating reagents cation patterns of redox sensors will assist in the further such as iodoacetamide, iodoacetic acid (IAA) and N-ethylmaleiclarification of the roles that redox sensors play in cancer cells, mide (NEM), all of which are commercially available, due to and may pave the road for the development of novel therapeu- its strong nucleophilicity [38]. IAA and IAM, which react with tic strategies for cancer therapy. thiolate via a nucleophilic substitution reaction to trap the Over the past few years, redox proteomics strategies based redox status of cysteine thiols, are by far the most commonly on thiol-trapping techniques have been developed for screening used alkylating reagents. However, IAA and IAM exhibit high new redox sensors and identifying the exact form of modifica- reactivity with nucleophilic side chains such as lysine tion. However, thiol-trapping strategies are still quite limited residues [39]. NEM can react with thiolate based on the O

doi: 10.1586/14789450.2015.1069189

Expert Rev. Proteomics


Screening cellular redox sensors

Michael-type addition. This reaction can be rapid enough to catch transient redox modification intermediates of proteins [34]. These alkylating agents are even accessible to free thiols in the interior of denatured proteins in the presence of sodium dodecyl sulfate (SDS). Although NEM exhibits rapid reaction kinetics with the thiolate of cysteines and has proven to be a relatively specific alkylating reagent, mis-alkylation of NEM has been observed on amines such as the imidazole of His and the "-amine of Lys [40]. To improve the specificity of the reaction between NEM and thiolates, reaction conditions (including the pH value, the concentration of NEM and the time of reaction) should be carefully optimized and monitored. It has also been reported that NEM can be used to quench thiols in intact living cells due to its permeability. However, a denaturant is required to enable all thiols to be exposed and accessible to NEM [41]. The reduced form of thiols can also be labeled by thiol-alkylating compounds (e.g., NEM, IAM, IAA) which are chemically conjugated with radiophores, biotin or fluorophores as reporter moieties. [42]. Additionally, polyethylene glycol (PEG) attached with maleimide (PEG-Mal) can also be used to label free thiols [43]. In this case, the oxidative state of proteins will be discriminated by their different molecular weights: proteins in which thiols are modified with PEG-Mal will exhibit slower mobility shift due to an increase in molecular weight due to the PEG [44]. The mobility shift depends on the number of PEG-Mal-labeled thiols. Different cysteine oxoforms, such as sulfenic acid, S-nitrosylation, and S-glutathiolation, hold different potential for regulating the redox sensor activity. A range of reagents designed for labeling different oxoforms of thiols have been developed [34]. Sulfenic acid can react with dimedone (5, 5-dimethyl-1, 3-cyclohexanedione) to form a stable thioether adduct [45,46] allowing dimedone to be used to specifically label sulfenylated cysteine (Cys-SOH). MS is used to identify the dimedone tags due to the lack of specific affinity tags [47]. Fluorescent and biotin labeling strategies can also be coupled with dimedone to enrich or identify sulfenic acid-containing proteins [47,48]. Another electrophilic reagent, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), has also been widely used to detect the sulfenic acid form of cysteines [38,49]. The bioorthogonal reactions of S-nitrosothiols (SNO) may selectively target the SNO. Thus a series of organophosphine compounds have been developed to label the SNO, specifically. For example, triaryl-substituted phosphines can react with the SNO rapidly to form an azaylide intermediate. This reaction is strictly limited to SNO moieties, rendering it a promising method to detect the S-nitrosylation of proteins [50]. Biotinylated glutathione ethyl ester (BioGEE) has been used to detect the S-glutathiolation of cysteines. Briefly, the BioGEE is made up by mixing EZ-Link sulfo-N-hydroxysuccinimidyl (NHS)-biotin and GSH ethyl ester in the presence of NaHCO3 at pH 8.5. NH4HCO3 is then added to remove excess biotinylation reagent. Subsequently, samples are incubated with the BioGEE, passed through a PD-10 Sephadex-G25 column and mixed with streptavidin-Sepharose beads. After elution from the informahealthcare.com

Review

streptavidin-Sepharose beads, S-glutathiolation of cysteines can be identified by Western blot [51,52]. Thiol enrichment strategies Biotin switch assay

In addition to the aforementioned thiol-labeling tags that are used for trapping reduced free thiols, the biotin switch assay capturing the oxidized form of thiols has also been developed. In this protocol, all of the free thiols are initially blocked using an alkylating reagent such as NEM, IAM, methyl methanethiosulfonate, or IAA. Oxidized thiols are then reduced using appropriate reductants to detect different patterns of thiol oxidation [53]. For example, arsenite can be used to specifically reduce sulfenylation [54], whereas glutaredoxin (Grx) triggers the reduction of cysteine S-glutathionylation [26]. Next, biotinylated reagents (e.g., biotinylated IAM (BIAM), biotinylated NEM, biotinylated maleimide, biotinylated N[6-(biotinamido) hexyl]-3’-(2’-pyridyldithio) propionamide (biotinylated HPDP), biotinylated iodoacetamidofluorescein) are incubated to react with newly formed free thiols (FIGURE 2). The very high affinity of streptavidin/avidin with biotin (dissociation constant 10 14 M) renders it feasible to readily enrich and purify the biotin-labeled proteins. Collectively, using the biotin switch assay, the intracellular status of oxidized thiols can be accurately monitored using appropriate biotinylated standards, and this labeling procedure can also be used in the shortgun proteomics approach [55]. In addition, by combining this protocol with mass spectrometry (MS), exactly which cysteine is oxidized can be identified [56]. The biotin-tagged methods have several advantages over other tags: the high affinity of streptavidin beads with biotin overcomes possible perturbation of the binding of antibody with redox-modified proteins; resins for enriching biotinylated proteins are readily available; and quantification can be achieved by SDS-PAGE followed by Western blotting or fluorescence imaging [57]. However, on the negative side, the biotin switch assay is labor intensive and not readily adapted to automation. Using modified biotin switch assays, Li and colleagues found that platelet releasates could trigger the accumulation of ROS through NADPH oxidase in an atherosclerosis model. In total, 75 oxidized peptides from 53 proteins were quantified. The enzymatic activity of several metabolic enzymes, such as GAPDH, triose phosphate isomerase along with a-enolase, was found to be inhibited via reversible oxidation of cysteine, which is consistent with previous reports [58]. As platelet releasates showed inhibitory effects upon multiple cellular activities (notably glycolysis), monitoring redox modification of proteins involved in various biological process is required to elucidate the role of oxidative stress in the development of disease [59]. Another method based on biotin has also been used to detect the redox status of cysteine residues [60]. Cell lysates were directly incubated with BIAM to trap the reduced form of thiols. In another workflow that shares similar concepts to the biotin switch assay, reduced samples can be blocked by the alkylating reagent and subsequently labeled with BIAM (FIGURE 3). doi: 10.1586/14789450.2015.1069189

Review


HS Ascorbate

ONS

ONS HS


GS-S

SOH

ONS

GS-S

GS-S

S-SR SH

ONS

MeSS

S-SR S-Biotin

MeSS

Biotin pull-down Western blotting

SOH

MMTS MeSS S-SR

SOH

MeSS

MeSS GS-S

Arsenite

Biotin-S

SOH

S-SR DTT

GS-S

S-SR

ONS

SOH

ONS Grx

GS-S

S-SR

ONS

SOH

MeSS

MeSS HS

Cell lysates

Biotin-HPDP

SH SOH

Biotin -S ONS

S-Biotin

Mass spectrometry

SOH

MeSS

MeSS HS

S-SR

Biotin-S

S-SR

Figure 2. The biotin switch assay workflow. Free thiols of cell lysates that possess diverse oxidative modifications are blocked by MMTS. Subsequently, oxidized thiols are reduced by suitable reductants. Third, Biotin-HPDP is incubated to react with newly formed free thiols. After enrichment, the oxidative modifications are identified using Western blotting (using antibodies such as anti-biotin or specific antibodies towards the protein of interest) or MS. DTT: Dithiothreitol; Grx: Glutaredoxin; HS: Hydroxysuccinimidyl; MMTS: Methyl methanethiosulfonate.

Resin-assisted enrichment of thiols

Thiol hydrophilic resins have been used as a thiol donor [61,62]. The resin-assisted thiol enrichment technique is based on the thiol-disulfide exchange (TDE) reaction with a thiol-affinity resin (e.g., thiopropyl sepharose 6B) and has been used as an effective and reproducible method to capture cysteinecontaining proteins [63,64]. Compared to the biotin switch assay that enriches thiols through biotin-avidin affinity, the resinassisted approach has the following advantage: the resin-assisted strategy uses direct trapping of cysteine-containing proteins in a thiol-affinity resin-dependent manner. On-resin tryptic digestion and isobaric (e.g., iTRAQ or tandem mass tags [TMT]) or stable isotope labeling are subsequently conducted without further sample clean-up prior to MS analysis. In addition, thiol-affinity resins capture cysteine-containing proteins through covalent reactions, which offer higher specificity and better sensitivity for thiol enrichment (>95% of Cys-containing peptides present in the final sample) [65]. However, there are still limitations in the resin-assisted approach: it is almost inevitable that false positives will occur as non-specific reductants are used. Thus, a carefully designed negative control is required for identification of artificial modifications [66]. In addition, detecting low-abundance reversible redox modifications by the resinassisted approach may be inefficient [67]. The method is very labor intensive, requiring approximately 3 day for sample processing with an additional day for LC-MS/MS and data analysis. Forrester’s group developed a method to specifically identify SNO based on resin-assisted capture (RAC), termed SNO-

doi: 10.1586/14789450.2015.1069189

RAC [63]. Later, Kohr’s group described another method termed Ox-RAC that is based on the previously reported SNO-RAC [68]. In this method, cell lysates are initially treated with ascorbate to remove SNO. The samples are then incubated with NEM to block free thiols. Subsequently, samples are incubated with selective reductants and added to thiopropyl Sepharose. Resin-bounded proteins are then digested with trypsin and finally analyzed by LC-MS/MS. Using Ox-RAC, Kohr’s group identified 158 proteins with oxidative modifications when samples are subjected to perfusion, IPC (ischemic preconditioning), IR (ischemia/reperfusion), and IPC-IR [68]. Redox proteomics for the global screening of cellular redox sensors Gel-based methods to identify redox sensors Non-reducing gel electrophoresis

Non-reducing gel electrophoresis is probably the most convenient method for the preliminarily investigation of the oxidative status of proteins in vivo or in vitro, as the formation of disulfides or other oxidative modifications gives rise to an alteration in mobility shift. Protein extracts prepared in nonreducing buffer are first blocked by alkylating reagents (IAM or NEM) and subsequently monitored by SDS-PAGE. Western blotting is then used with specific antibodies to detect the oxidation state of the proteins. However, in addition to adjacent free thiols, which can form intramolecular or intermolecular disulfides thus altering the molecular mass, thioredoxin (Trx), Prx or other oxidoreductases can also potentially bind with an oxidative thiol, resulting in a shift in gel mobility [69].


Review



Therefore, the combination of nonreducing gel with MS analysis is necessary to discern whether the change in molecular mass is due to the formation of disulfides. As an example, KIADH1, an alcohol dehydrogenase isozyme in Kluyveromyces lactis, forms an inactive tetramer (150kDa) in yeast. The redox pool of its cysteines has been explored as the activity of KIADH1 is notably affected by reductants [70]. Reducing and non-reducing gels were used to analyze the change of molecular mass of KIADH1 in response to treatment with disulfide-inducers or reductants. As a result, Cys278 was shown to be involved in the formation of a disulfide leading to the reversible inactivation of KIADH1 [71]. Diagonal gel electrophoresis

A P1

B

SH

P2

SX

P2

Protein solution

SH

BIAM labeling

P1

S-IAM-Biotin

P1

SX

Protein solution

P2

NEM

P2

SX

S-NEM

P1

SX

P1

SH

TCEP Streptavidin Agarose beads P1

P2

S-NEM

S-IAM-Biotin-Streptavidin-beads P2

BIAM labeling

SX SDS-PAGE & WB + anti-P1 antibody

HRP-antibody

P1

P2

S-IAM-Biotin

SDS-PAGE & WB + anti-P1 antibody

S-NEM

P1

S-IAM-Biotin

Streptavidin Agarose beads P1

S-IAM-Biotin-Streptavidin-beads

Disulfide formation can fall into two P2 S-NEM major types: intramolecular or intermolecular disulfide bonds. Diagonal gel elecFigure 3. An overview of the BIAM-labeled method. (A) Free thiols are directly trophoresis was developed in the 1970s incubated with BIAM, streptavidin agarose beads are subsequently used to conjugate as a relatively simple method to discrimiwith biotin. Finally, SDS-PAGE and Western blotting analysis are conducted to identify nate both intra-molecular and interthe oxidative state of proteins. (B) Reduced thiols are first blocked by alkylating reagents, reductants are subsequently added to reduce reversible oxidized thiols, and molecular disulfides [72]. Protein extracts newly formed free thiols are labeled by BIAM. The subsequent procedures are identical are prepared in non-reducing buffer and to A. The oxidative state of proteins can be indirectly identified by this approach. then separated by SDS-PAGE (nonBIAM: Biotinylated iodoacetamide; NEM: N-ethylmaleimide; SDS: Sodium dodecyl sulfate. reducing dimension). The corresponding lanes are then excised and incubated in dithiothreitol or 2-mercaptoethanol to reduce all disulfides. SYPRO Ruby dye. The spot of interest is then excised from Following incubation, the gels are rotated through 90 and run the gel and analyzed by MS [77]. Software such as DECODON as stacking gel for SDS-PAGE (reducing dimension). Proteins Delta 2D can be used for 2-DE quantitative image analysis. without disulfides run identically in both reducing and non- Western blotting can be combined with 2-DE to improve both reducing dimensions thus forming a diagonal. Proteins with the specificity and sensitivity. Combined with enrichment techintermolecular disulfides are identified below the diagonal as a niques, 2-DE-MS and shotgun-MS may complement each result of molecule mass reduction under reductant treatment, other to characterize diverse forms of oxidation modificawhereas proteins with intra-molecular disulfides run above the tions [78]. However, multiple proteins can co-migrate in 2-DE, diagonal as the protein conformation becomes linear when the which means absolute quantification may not be possible [79]. disulfides are reduced [73,74]. The protein spot of interest can In addition, hydrophobic proteins or proteins with high isothen be excised from the gel, digested with trypsin, and ana- electric point may be poorly resolved by 2-DE, although many lyzed by MS. Diagonal gel electrophoresis has been used to of the earlier perceived limitations of 2-DE have now been identify the disulfide bond pattern of certain purified redox either effectively addressed or established to be little more than dogma associated with poor sample handling and methodologisensors [75]. cal practices. However, 2-DE per se provides little information about the specific cysteines involved in disulfide formation and Two-dimensional electrophoresis Two-dimensional electrophoresis (2-DE) is a gel-based method, the inherent poor reproducibility of 2-DE makes it difficult to which can be used to detect the different expression of protein accurately compare samples run on different gels [42]. Redox fluorescence switch (RFS) was recently developed following various treatments [76]. For example, samples treated with hydrogen peroxide (H2O2) or diamide when applied to by Alicia and colleagues to label reversibly oxidized thiols 2-DE separate depending on two key characteristics, the iso- with a fluorophore. Following 2-DE separation, numerous electric point and molecular mass of the proteins. After separa- redox-sensitive proteins were identified involved in physiologition, gels are stained with Coomassie brilliant blue G-250 or cal responses to hypoxia [80]. informahealthcare.com

doi: 10.1586/14789450.2015.1069189

Review



Two-dimensional difference gel electrophoresis

Two-dimensional difference gel electrophoresis (2D-DIGE) was developed to overcome the poor reproducibility of 2-DE, where different protein samples can be labeled with size and charge matched spectrally resolvable fluorescent dyes, allowing two or more samples to be detected simultaneously in the same gel using differential fluorescence wavelength scanning. Fluorescent dyes such as N-hydroxysuccinimidyl (NHS) ester derivatives of Cy2, Cy3, and Cy5 (NHS-Cy2, NHS-Cy3, NHS-Cy5) are commonly used fluorescent labeling reagents [81]. 2D-DIGE is sensitive enough to detect as little as 0.5 fmol (femptomole) of samples and has a 10,000-fold dynamic range. Redox-DIGE [82] can be used to distinguish different redox states of proteins. In this procedure, free thiols are blocked by NEM; then protein extracts are treated with selective reductant and subsequently labeled with Cy5-maleimide or Cy3-maleimide. The protein extracts are then pooled and resolved by 2-DE on one gel. Finally, the fluorescence is scanned at different wavelengths giving red or green spots representing different patterns of redox modifications. Despite the relatively high reproducibility and sensitivity of 2D-DIGE, a noted caveat is that 2-DE is not efficient at resolving integral membrane proteins due to their precipitation during isoelectric focusing (IEF). More recently, ‘click chemistry’ (Cu1+-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC) reaction) has been combined with 2D-DIGE and applied to redox proteomics [83]. Homopropargylglycin, an analog of methionine, can react selectively with Cys3 and Cys5 on their azide groups using the CuAAC reaction, giving a novel way to identify redox-sensitive proteins [83]. In a study of UVB-induced skin cancer, 2D-DIGE was applied to measure the differential expression and redox state of multiple proteins. Thirty-seven proteins were identified with different redox thiol modifications associated with tumorigenesis [84]. In another study, redox 2D-DIGE was applied to identify proteins with altered redox modification in PRDX1 knockdown cells. In total, 30 proteins were recognized as redoxmodified candidates, the majority of which participate in cell death and cellular stress response pathways, suggesting that multiple redox sensors involved in the cellular network are potential targets for enhancing the curative effect of chemotherapeutics [85]. In addition, using 2D-DIGE, Chan’s group has demonstrated that when glutathione reductase (GR), an enzyme which plays a vital role in modulation cellular redox homeostasis, was knocked down, several proteins including Prx-1 were differentially oxidized and had alternative protein expression in human lung cancer cells [86]. Future studies on redox-sensitive proteins could unveil the roles different cysteine oxoforms of redox sensors play in lung cancer initiation and progression. In another study based on breast cancer, 2D-DIGE was combined with MS to investigate the berberine induced redox regulation on proteins in MCF-7 cells. Twenty-two proteins were reported to alter their redox status and 17 thiol sites with alternative redox status on nine proteins were inferred [87]. Possible roles of these redox sensitive proteins in berberine-induced cytotoxicity need further investigation. doi: 10.1586/14789450.2015.1069189

The major strength of gel-based approaches is that these methodologies can be routinely monitored. Results are easy to acquire and analyze in contrast to the complex data obtained from MS analysis, which is data intensive. Moreover, 2D-DIGE allows multiple samples to be separated on the same gel, which overcomes the gel-to-gel reproducibility problems observed in simple 2-DE. In general, gel-based methods are still required to minimize intrinsic limitations such as the artificial oxidation occurring during sample collection and purification. However, these methodologies are only semi-quantitative and lack sensitivity and specificity. Importantly, the specific site of cysteine residues involved cannot be identified using gelbased methods alone. Antibody-based methods to detect the oxidative state of thiols

Using specific antibodies to monitor the oxidation form of cysteine residues offers a straightforward method of detection of the redox status of protein, and antibodies have been developed to scout for the oxidative state of free thiols, including S-glutathionylation and S-sulfenylation [88,89]. For example, a pan-specific antibody was produced in Maller’s group that can specifically detect sulfenylation (Pr-SOH) under dimedone treatment [90]. In addition, free thiols can be modified by reactive lipids such as malondialdehyde, nitrated fatty acids, and 4-hydroxynonenal. Broad-spectrum antibodies are then developed against malondialdehyde- or the nitrated fatty acidsmodified proteins [91]. However, antibodies may not identify all redox modifications due to the limited recognization of specific epitopes. Antibodies designed to detect sulfinated and sulfonated forms have been developed, and consequently, sulfinylation (Pr-SO2H) and sulfonylation (Pr-SO3H) in cysteinecontaining proteins can be recognized [92]. Antibody-based methods can also be used to purify proteins with specific oxidative modifications. However, proteins without reactive thiols can also be pulled down in the purified mixture as other proteins can form complexes with redox-sensitive proteins [44]. Gel-free methods to identify redox pools

Due to the inherent limitations of gel-based methods, strategies based on HPLC were subsequently developed with the same general concept: modification of disulfide bonds-containing proteins by an appropriate reductant confers the protein with different mobility properties and an altered elution time, through which the oxidation state of the protein can be estimated [93]. Similarly, HPLC combined with downstream MS analysis offers additional information about the specific sites of disulfide bonds. MS-based methods

Recent rapid developments in MS analysis have resulted in better sensitivity and specificity that enable redox proteomics to uncover new redox sensors present at low abundance. Free thiols in protein extracts are labeled with a suitable alkylating reagent. Subsequently, the reversibly oxidized thiols are reduced Expert Rev. Proteomics



by reductants and further incubated with another alkylating reagent. The labeled-protein extracts are then typically separated by SDS-PAGE, digested with trypsin, and subjected to the LC-MS/MS analysis. Using this approach, reduced and oxidized form of cysteines can be simultaneously identified as different alkylating reagents can be differentiated by MS [94]. In addition to identify reversible oxidative modifications, a scheme to isolate sulfinylated (Pr-SO2H) and sulfonylated (Pr-SO3H) thiols has been developed [95]. Using this method, 181 CysSO2H/ SO3H peptides were identified after ischemia/ reperfusion injury in rat myocardial tissue [95]. To quantify oxidized cysteines, stable isotope labeling of oxidized thiols has been developed. In detail, free thiols are initially blocked by alkylating reagents. Reductants are then used to reduce the oxidized thiols and the newly-formed thiols are labeled with a stable isotope [96]. However, this method does not include a protein purification step and cannot be applied to diverse samples simultaneously. Moreover, the ratio of unmodified thiols and oxidized thiols is not achievable as the free thiols are not labeled. Recently, a shotgun-LC-MS/MS-based method was developed to characterize thiol modifications [97]. In this protocol, protein extracts were resuspended in urea/CHAPS alkylation buffer containing IAM to label free thiols. Subsequently, reversibly oxidized thiols were reduced by tris-(2-carboxyethyl) phosphine (TCEP), and nascent free thiols were labeled with BODIPY FL C1-IA [N-(4, 4-difluoro-5, 7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-yl)-methyl]-iodoacetamide]. The fluorescently labeled protein sample was then applied to 2D PAGE for separation, as described above. Finally, the gel was scanned using a Typhoon imaging system (GE Healthcare) and costained with Colloidal Coomassie for protein quantitation. Spots were excised and digested with trypsin for MALDI-TOFTOF analysis. In addition to shot-gun proteomics, top-down proteomics strategies are rapidly developed that allow the unequivocal identification and location of specific post-translational modifications [98]. More recently, the top-down approaches have been applied to the detection of redox modifications such as disulfide bonds analysis and S-nitrosylation [99,100]. As enzymatic digestion is not required for top-down approaches, enabling the more detailed identification of post-translational modifications than shot-gun proteomics, top-down proteomics might hold the potential to be an alternative and improved method of identifying the redox status of protein thiols. ICAT-based technology

An alternative approach based on isotope-coded affinity tag (ICAT) technology has also been developed with a view of comparing the pattern of oxidized thiols in paired samples (FIGURE 4) [101,102]. Specifically, cell lysates from samples that have undergone different treatments are incubated with light or heavy ICAT reagents. The protein mixture is then digested by trypsin and fractioned by HPLC followed by MS/ MS analysis. Using this proteomic technique, Cohen’s group informahealthcare.com

Review

found that Cys 278 of sarcomeric creatine kinase is highly reactive and susceptible to oxidative modification [103]. Another efficient ICAT-based method has been described by Hidago’s group. In this protocol, TCA is initially added to cell lysates to protonate all of the redox-active thiolate anions and quench thiol-disulfide exchange (TDE) by changing the pH as well as denaturing and precipitating proteins. Free thiols are blocked with IAM, reversibly oxidized thiols are then reduced by selective reductants and subjected to biotinylated light ICAT (B-12C-ICAT) or heavy ICAT (B-13C-ICAT). Following isotope labeling, the samples are mixed up in equal ratios and digested by trypsin. Samples are finally purified with avidin and analyzed by LC-MS/MS [104]. Nevertheless, this method merely provides the ratio of reversible oxidized thiols in different samples, and the global abundance of proteins is not revealed. It is important to quantify the relative expression of proteins as treatment may alter both expression levels and the oxidative modification. To improve the quantification of protein expression versus the reversible oxidized thiols, label-free or stable isotope-based strategies can be applied to the quantification of non-enriched peptides [105]. Dimethyl labeling, TMT and isobaric tags for relative and absolute quantification (iTRAQ) are viable strategies widely used for relative protein quantification [106,107]. Using the optimized ICAT-based method, Tpx1 and Pap1 were identified as redox-sensitive proteins involved in yeast fission [101]. CysTMTRAQ is another novel method combining TMT and isobaric tags for quantification, thus allowing the simultaneous analysis of the redox states of thiols and the global protein level [104,108]. In order to both identify variations of protein expression and analyze the percentage of redox modification, Jackson’s group have reported a label-free quantitative proteomic approach that includes a differential cysteine labeling step [109]. Using this method, individual proteins are quantified. Thus changes of the oxidative modifications can be detected with information on the overall protein abundance. Jacob’s group has also developed an ICAT-based method, OxICAT, to identify the redox status of proteins (FIGURE 4). Samples are initially acidified with TCA to maintain the redox status of proteins. Reduced thiols are then labeled with light ICAT (B-12C-ICAT), and reversibly oxidized forms of thiols are reduced by selective reductants (e.g., TCEP) and labeled by a 9-Da-heavier isotopically heavy form (B-13C-ICAT) [110]. Substitution of TCEP with more selective reductants such as glutaredoxin (Grx) will enable the identification of protein glutathionylations using OxICAT. OxICAT has been successfully used to compare the relative abundances of reduced and oxidized thiols, which could further advance our understanding of global redox modifications of cysteines [111]. In addition, OxICAT was shown to be a sensitive, quantitative and site-specific proteomic approach to investigate the redox proteome (FIGURE 4) [101]. OxICAT has been successfully used to monitor the oxidative state of cysteine-containing proteins at different time points in the C. elegans lifespan [112]. These data showed that more doi: 10.1586/14789450.2015.1069189


12C 13 C

12C

Sample A OxICAT

12C

S-R

ICAT

S- 12C

SH

13C

ICAT

S- 13C 12 S- C

TCEP

S

Digest Purification

s

Intensity [AU]

S-R

LC/MS

13C

S

S-R

SH

12C

S-IAM S-R


SH

13C

S-IAM

ICAT

S-IAM Digest S- 13C

TCEP

LC/MS

Purification

S-IAM

Dimethyl labeling based relative protein quantification D-CDO

LC/MS

D-13CDO

S-NEM

S-H

S-R

S-NEM S-R

SH

Biotin-HPDP

114 S

Digest Purification

S-biotin

S

MS/MS

118 S

S-biotin

S-NEM

13CD 6

116

iTRAQ reagents

S-NEM

S-R

SH

m/z

S-NEM

DTT

S-NEM

S-R

12CD O 4 2

S-biotin

NEM S-R

m/z

S-NEM

S-R

SH

s 9Da

S-NEM

S-NEM

SH

s

13 CD 6

Digest

Sample C OxiTRAQ

m/z

12C 13 C

S- 12C

TCEP

IAM S-R

ICAT

Intensity [AU]

S-R

ICAT-based method

Intensity [AU]

Sample B

s 9Da

Intensity [AU]

Review

12CD O 4 2

m/z

121

S-NEM S

S-R

Sample D OxMRM

SH

d0NEM

S-R S- d0NEM

S-H

TCEP

S- d0NEM

d5NEM

S-d5NEM S- d0NEM

LC-MS/MRM Digest Purification

d0NEM

d5NEM

Figure 4. Schematic illustration of OxICAT, ICAT-based method and OxiTRAQ. (A) Reduced thiols are labeled with light ICAT (12C-ICAT), new thiols reduced by TCEP are labeled using a 9-Da-heavier isotopic form (13C-ICAT). The ratios of oxidized and reduced thiols are then identified by LC/MS analysis. (B) Free thiols are labeled with IAM, reversible oxidized thiols are then reduced by TCEP and subjected to 12C-ICAT or 13C-ICAT. The oxidative state in different samples is analyzed by LC-MS/MS. Finally, the global abundance of proteins is quantified using dimethyl labeling strategies. (C) Proteins extracts of different samples are incubated with NEM to block free thiols. Subsequently, reversible oxidative modifications are reduced and labeled with biotin-HPDP. Isobaric iTRAQ reagents are incubated with biotin-captured peptides and finally MS/MS analysis is applied to identify the oxidative state of different samples. (D) Protein extracts are incubated with d0 NEM to block initial free thiols, and then reductants are added to reduce cysteine oxoforms. Subsequently, d5 NEM is used to label the nascent thiols. Finally, digested samples are applied to LC-MS/MRM analysis. DTT: Dithiothreitol; ICAT: Isotope-coded affinity tag; IAM: Iodoacetamide; iTRAQ: Isobaric tags for relative and absolute quantification; LC: Liquid chromatography; MRM: Multiple reaction monitoring; MS: Mass spectrometry; NEM: N-ethylmaleimide.

oxidative thiols were present in aged C. elegans and worms. Proteins including HSP-1 (Hsp70 homologue), MEL-32, the protease neprylisin, and UNC-87 were highly oxidative during C. elegans development, most of which were distributed ubiquitously and have been previously reported as peroxidesensitive [113]. However, costly ICAT reagents are required for the detection of both oxidized thiols and reduced thiols. In addition, the low abundance of purified cysteine-containing peptides renders it difficult to analysis by MS, making the data analysis of OxICAT even more complex [104,114]. doi: 10.1586/14789450.2015.1069189

OxiTRAQ

OxiTRAQ was developed to detect the redox modifications of multiple samples simultaneously [115]. Proteins extracts of different samples are incubated with NEM to block reduced thiols. The reversible oxidative modifications are then reduced and labeled with biotin-HPDP. The biotinylated proteins are purified by avidin-affinity chromatography and digested by trypsin. Subsequently, isobaric iTRAQ reagents with various isotopecoded tags (e.g., reporter 114, 116, 118, 121) are incubated with the biotinylated-peptides [116]. Finally, peptides with multifarious tags are mixed and analyzed by MS/MS analysis.



Compared with OxICAT, OxiTRAQ is capable of analyzing more than one sample in a single run. Additionally, compared with TMT, iTRAQ is more sensitive to cysteine-containing peptides and allows more samples to be quantified simultaneously [117,118]. In short, the OxiTRAQ is a sensitive, quantitative and site-specific proteomic method to investigate the redox proteomes.


OxMRM

Gibson’s group has developed a highly efficient approach, termed OxMRM (quantitative cysteine oxidation analysis by multiple reaction monitoring), which combines the differential alkylation of samples with stable isotopes and MRM to analysis the oxidative status of cysteines [94]. OxMRM is characterized as a sensitive and unbiased targeted assay, allowing proteins with low-abundance to be quantified. Briefly, TCA is added to the cell lysates to minimize artificial oxidation. Unlabeled (d0) NEM is then combined with 8 M urea and 2% (w/v) SDS to block the free thiols. Subsequently, selective reducing reagents, such as DTT, TCEP, arsenite or ascorbate, are used to reduce different oxoforms of cysteines. Nascentfree thiols are then labeled by NEM with 5 deuteriums (d5). Finally, the protein of interest is digested and applied to LC-MS/MRM. MRM protocols can be developed to distinguish between d0 and d5 labeled peptides and thus quantify the percentage of oxidation. In this way, OxMRM can be used to identify the various oxidative states of cysteines. Importantly, when a certain peptide undergoes multiple oxidative modifications, OxMRM is capable of measuring the cysteines involved in a single run [34]. Using OxMRM, Gibson’s group found that the Cys 182 of p53 (a low-abundance protein) could be oxidized following exposure to diamide. As Cys 182 lies in the interface of p53 dimers, oxidized Cys 182 may exert effects on the formation of tetramer and ultimately influence the binding of p53 and DNA [94]. OxMRM provides a robust quantification of unbiased proteomics approaches and is sensitive to detect the established oxidative modification of cysteines. pLink-SS-based method to map disulfide bonds

pLink, a software aimed at analyzing data for chemically cross-linked proteins, was developed by Yang and colleagues and has been applied to detect protein–protein interactions and protein folding [119]. The software was also adapted to enable mapping of disulfide bonds within numerous proteins, termed ‘pLink-SS’ [120]. Using pLink-SS along with MS analysis, 199 disulfide bonds in 150 proteins of E. coli have been identified by comparing disulfide proteomes in dsbA , dsbC (DsbA and DsbC are thiol-disulfide oxidoreductases) and wild-type BW25113 strains [121]. In addition, 309 disulfide bonds and 1738 glutathionylated sites were identified in A549 cells under diamide treatment. Moreover, when monitoring the distance of two cysteine residues forming disulfide bonds, they found that CXXC is the most prominent mode in disulfide bonds-formation in E. coli, and the formation of informahealthcare.com

Review

disulfide bonds could occasionally alter protein conformations by expelling metal ions [120]. Disulfide proteome analysis provides new concepts for exploring redox-sensitive proteins and mapping disulfide bonds critical for protein function. Using redox proteomics approaches, a mass of redoxsensitive proteins have been identified in distinct types of cancer as mentioned above. The major focus of further studies should be on the alternative biological functions of different cysteine oxoforms in individual protein to obtain novel targets in cancer therapy. In summary, the use of redox proteomics to investigate redox status alterations of protein cysteine residues in cancer may constitute a comprehensive approach to elucidate the underlying mechanism by which ROS promote cancer initiation and progression. Expert commentary

ROS was initially considered as an ‘antihero’ that would lead to damage of cellular biomolecules and finally contribute to the development of disease [122]. As increasing numbers of proteins modified by ROS were verified to modulate cellular events, including apoptosis, autophagy, cell cycle and metabolism, the role of ROS became ambiguous [92]. Several oxidative modifications in cysteine-containing proteins could change the inherent function of proteins that are involved in cellular inflammatory response (e.g., NF-kB) [123] and tumorigenesis (e.g., pyruvate kinase M2) [19]. Hence, the development of a comprehensive redox proteomics strategy is urgently required for mapping global oxidative modifications. Collectively, redox proteomics based on thiol-trapping strategies could enable the screening of redox sensors holding the potential to be therapeutic targets for cancer. Five-year view

Understanding the redox state of proteins involved in tumorigenesis may bring with it the potential to identify new therapeutic targets for cancer. Recent advances in gelbased or non-gel based approaches have contributed to advances in redox proteomics in identifying cysteine modifications during cancer development. Many methods have been proposed for the detection of redox sensors. However, potential limitations still exist, including artifactual oxidation, poor reproducibility, inefficient identification of redox sensors with low abundance, lack of suitability for highthroughput development as well as a lack of multiplex labeling techniques. Numerous studies have been performed to overcome these inherent defects. Developing novel thioltrapping strategies to evade artificial oxidation during sample preparation will significantly improve method reproducibility. Optimistically, with the development of such improved thiol-trapping strategies, MS techniques and software analysis in the next decade, viable methods with higher sensitivity and reproducibility will be developed that will benefit the global identification of redox modifications of pivotal proteins and facilitate further understanding of redox-regulated tumorigenesis. doi: 10.1586/14789450.2015.1069189

Review


Financial & competing interests disclosure

C Huang was supported by grants from the National 973 Basic Research Program of China (2011CB910703, 2013CB911300 and 2012CB518900), the Chinese NSFC (81225015 and 81430071), and Sichuan Science-Technology Innovative Research Team for

Young Scientist (2013TD0001). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Key issues .

Reactive oxygen species induce the redox modification of a range of proteins involved in tumor development.

.

Thiol-trapping methods such as acidification and thiol-labeling are widely used, and are pivotal for quenching the redox state prior to sample preparation.


.

Both the biotin-switch assay and resin-assisted enrichment are reported to be feasible schemes for the enrichment of cysteinecontaining proteins.

.

Both gel-based methods and MS-based methods aimed at identification of redox pools have been developed.

.

Artificial oxidation and poor reproducibility are significant bottlenecks in redox proteomics.

.

The development of high-throughput protocols compatible with large-scale proteome analysis may require a move away from gel-based techniques.

.

The pLink-SS bioinformatics software (derived from Plink) will assist in the analysis of protein disulfide bonds.

Papers of special note have been highlighted as: . of interest .. of considerable interest 1.

Cairns RA, Harris IS, Mak TW. Regulation of cancer cell metabolism. Nat Rev Cancer 2011;11(2):85-95

2.

Kuo MT. Redox regulation of multidrug resistance in cancer chemotherapy: molecular mechanisms and therapeutic opportunities. Antioxid Redox Signal 2009; 11(1):99-133

3.

Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013;12(12):931-47

4.

Oberley LW. Free radicals and diabetes. Free Radic Biol Med 1988;5(2):113-24

5.

Wen X, Wu J, Wang F, et al. Deconvoluting the role of reactive oxygen species and autophagy in human diseases. Free Radic Biol Med 2015;65:402-10

6.

Aschner M, Caito SW. Mitochondrial redox dysfunction and environmental exposures. Antioxid Redox Signal 2015. [Epub ahead of print]

7.

Yang W, Zou L, Huang C, Lei Y. Redox regulation of cancer metastasis: molecular signaling and therapeutic opportunities. Drug Dev Res 2014;75(5):331-41

8.

Hensley K, Harris-White ME. Redox regulation of autophagy in healthy brain and neurodegeneration. Neurobiol Dis 2015; Epub ahead of print

9.

Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for

doi: 10.1586/14789450.2015.1069189

18.

Voth W, Schick M, Gates S, et al. The protein targeting factor Get3 functions as ATP-independent chaperone under oxidative stress conditions. Mol Cell 2014;56(1): 116-27

19.

Anastasiou D, Poulogiannis G, Asara JM, et al. Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses. Science 2011; 334(6060):1278-83

20.

Miyata Y, Rauch JN, Jinwal UK, et al. Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70. Chem Biol 2012;19(11):1391-9

21.

Antico Arciuch VG, Galli S, Franco MC, et al. Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression. PLoS One 2009;4(10): e7523

22.

Roeser J, Bischoff R, Bruins AP, Permentier HP. Oxidative protein labeling in mass-spectrometry-based proteomics. Anal Bioanal Chem 2010;397(8):3441-55

Cooper CE, Patel RP, Brookes PS, Darley-Usmar VM. Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species. Trends Biochem Sci 2002;27(10): 489-92

23.

Wang K, Zhang T, Dong Q, et al. Redox homeostasis: the linchpin in stem cell self-renewal and differentiation. Cell Death Dis 2013;4:e537

Wang Y, Yang J, Yi J. Redox sensing by proteins: oxidative modifications on cysteines and the consequent events. Antioxid Redox Signal 2012;16(7):649-57

24.

Shao D, Oka S, Liu T, et al. A redox-dependent mechanism for regulation of AMPK activation by thioredoxin1 during energy starvation. Cell Metab 2014;19(2):232-45

Jeong W, Bae SH, Toledano MB, Rhee SG. Role of sulfiredoxin as a regulator of peroxiredoxin function and regulation of its expression. Free Radic Biol Med 2012; 53(3):447-56

25.

Woo HA, Jeong W, Chang TS, et al. Reduction of cysteine sulfinic acid by

suicide and survival. J Cell Physiol 2002; 192(1):1-15

References 10.

Ranjan P, Anathy V, Burch PM, et al. Redox-dependent expression of cyclin D1 and cell proliferation by Nox1 in mouse lung epithelial cells. Antioxid Redox Signal 2006;8(9-10):1447-59

11.

Cobb CA, Cole MP. Oxidative and nitrative stress in neurodegeneration. Neurobiol Dis 2015; Epub ahead of print

12.

Brown DI, Griendling KK. Regulation of signal transduction by reactive oxygen species in the cardiovascular system. Circ Res 2015;116(3):531-49

13.

Vlahos R, Selemidis S. NADPH oxidases as novel pharmacologic targets against influenza a virus infection. Mol Pharmacol 2014;86(6):747-59

14.

15.

16.

17.

Oyinloye BE, Adenowo AF, Kappo AP. Reactive oxygen species, apoptosis, antimicrobial peptides and human inflammatory diseases. Pharmaceuticals (Basel) 2015;8(2):151-75



26.


27.

28.

29.

30. 31.

sulfiredoxin is specific to 2-cys peroxiredoxins. J Biol Chem 2005;280(5): 3125-8

38.

Peng H, Chen W, Cheng Y, et al. Thiol reactive probes and chemosensors. Sensors (Basel) 2012;12(11):15907-46

Shelton MD, Chock PB, Mieyal JJ. Glutaredoxin: role in reversible protein s-glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid Redox Signal 2005; 7(3-4):348-66

39.

Nielsen ML, Vermeulen M, Bonaldi T, et al. Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry. Nat Methods 2008;5(6):459-60

40.

Delaunay A, Pflieger D, Barrault MB, et al. A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell 2002;111(4):471-81 Lee TH, Kim SU, Yu SL, et al. Peroxiredoxin II is essential for sustaining life span of erythrocytes in mice. Blood 2003;101(12):5033-8 Heneberg P. Reactive nitrogen species and hydrogen sulfide as regulators of protein tyrosine phosphatase activity. Antioxid Redox Signal 2014;20(14):2191-209 Wang Z. Protein S-nitrosylation and cancer. Cancer Lett 2012;320(2):123-9 Nakamura T, Prikhodko OA, Pirie E, et al. Aberrant protein S-nitrosylation contributes to the pathophysiology of neurodegenerative diseases. Neurobiol Dis 2015; Epub ahead of print

32.

Haldar SM, Stamler JS. S-nitrosylation: integrator of cardiovascular performance and oxygen delivery. J Clin Invest 2013;123(1): 101-10

33.

Hansen RE, Winther JR. An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Anal Biochem 2009;394(2): 147-58

41.

Paulech J, Solis N, Cordwell SJ. Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry. Biochim Biophys Acta 2013; 1834(1):372-9 Lind C, Gerdes R, Hamnell Y, et al. Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch Biochem Biophys 2002;406(2):229-40

42.

Charles R, Jayawardhana T, Eaton P. Gel-based methods in redox proteomics. Biochim Biophys Acta 2014;1840(2):830-7

43.

Hoppe G, Talcott KE, Bhattacharya SK, et al. Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp Cell Res 2006;312(18):3526-38

44.

Eaton P. Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures. Free Radic Biol Med 2006;40(11):1889-99

Review

51.

Garcia-Gimenez JL, Olaso G, Hake SB, et al. Histone h3 glutathionylation in proliferating mammalian cells destabilizes nucleosomal structure. Antioxid Redox Signal 2013;19(12):1305-20

52.

Bachschmid MM, Xu S, Maitland-Toolan KA, et al. Attenuated cardiovascular hypertrophy and oxidant generation in response to angiotensin II infusion in glutaredoxin-1 knockout mice. Free Radic Biol Med 2010;49(7):1221-9

53.

Thamsen M, Jakob U. The redoxome: proteomic analysis of cellular redox networks. Curr Opin Chem Biol 2011; 15(1):113-19

..

This paper reviews the redox proteomics approaches for mapping cellular thiol state and provides a comprehensive prococol of biotin-switch assays.

54.

Saurin AT, Neubert H, Brennan JP, Eaton P. Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. Proc Natl Acad Sci USA 2004; 101(52):17982-7

55.

Wan J, Roth AF, Bailey AO, Davis NG. Palmitoylated proteins: purification and identification. Nat Protoc 2007;2(7): 1573-84

56.

Burgoyne JR, Eaton P. A rapid approach for the detection, quantification, and discovery of novel sulfenic acid or S-nitrosothiol modified proteins using a biotin-switch method. Methods Enzymol 2010;473:281-303

45.

Allison WS. Formation and reactions of sulfenic acids in proteins. Acc Chem Res 1976;9(8):293-9

57.

46.

Together with 93, these papers comprehensively describe the pros and cons of various thiol-trapping strategies.

Alvarez B, Carballal S, Turell L, Radi R. Formation and reactions of sulfenic acid in human serum albumin. Methods Enzymol 2010;473:117-36

Hill BG, Reily C, Oh JY, et al. Methods for the determination and quantification of the reactive thiol proteome. Free Radic Biol Med 2009;47(6):675-83

58.

47.

34.

Held JM, Gibson BW. Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes. Mol Cell Proteomics 2012;11(4):R111 013037

Carballal S, Radi R, Kirk MC, et al. Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite. Biochemistry 2003;42(33): 9906-14

Nakajima H, Amano W, Fujita A, et al. The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death. J Biol Chem 2007;282(36):26562-74

48.

Winther JR, Thorpe C. Quantification of thiols and disulfides. Biochim Biophys Acta 2014;1840(2):838-46

Poole LB, Zeng BB, Knaggs SA, et al. Synthesis of chemical probes to map sulfenic acid modifications on proteins. Bioconjug Chem 2005;16(6):1624-8

59.

35.

36.

Cortese MS, Baird JP, Uversky VN, Dunker AK. Uncovering the unfoldome: enriching cell extracts for unstructured proteins by acid treatment. J Proteome Res 2005;4(5):1610-18

49.

60.

Kim JR, Lee SM, Cho SH, et al. Oxidation of thioredoxin reductase in HeLa cells stimulated with tumor necrosis factor-alpha. FEBS Lett 2004;567(2-3):189-96

37.

Bensadoun A, Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem 1976;70(1):241-50

Denu JM, Tanner KG. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 1998;37(16):5633-42

Li R, Huang J, Kast J. Identification of total reversible cysteine oxidation in an atherosclerosis model using a modified biotin switch assay. J Proteome Res 2015; 14(5):2026-35

61.

Badyal JP, Cameron AM, Cameron NR, et al. A simple method for the quantitative analysis of resin bound thiol groups. Tetrahedron Lett 2001;42(48):8531-3

..


50.

Torta F, Elviri L, Bachi A. Direct and indirect detection methods for the analysis of S-nitrosylated peptides and proteins. Methods Enzymol 2010;473:265-80

doi: 10.1586/14789450.2015.1069189

Review 62.

Deratani A, Sebille B. Metal ion extraction with a thiol hydrophilic resin. Analy chem 1981;53(12):1742-6

73.

McDonagh B. Diagonal electrophoresis for the detection of protein disulfides. Methods Mol Biol 2012;869:309-15

63.

Forrester MT, Thompson JW, Foster MW, et al. Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat Biotechnol 2009;27(6):557-9

..

Together with 74, these papers comprehensively describe the protocol of diagonal gel electrophoresis for mapping cellular thiol state.

64.



65.

66.

67.

68.

69.

70.

71.

72.

Paulech J, Solis N, Edwards AV, et al. Large-scale capture of peptides containing reversibly oxidized cysteines by thiol-disulfide exchange applied to the myocardial redox proteome. Anal Chem 2013;85(7):3774-80 Su D, Shukla AK, Chen B, et al. Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry. Free Radic Biol Med 2013;57:68-78 Murray CI, Uhrigshardt H, O’Meally RN, et al. Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay. Mol Cell Proteomics 2012;11(2):M111 013441 Guo J, Gaffrey MJ, Su D, et al. Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications. Nat Protoc 2014;9(1):64-75 Kohr MJ, Sun J, Aponte A, et al. Simultaneous measurement of protein oxidation and S-nitrosylation during preconditioning and ischemia/reperfusion injury with resin-assisted capture. Circ Res 2011;108(4):418-26 Sobotta MC, Liou W, Stocker S, et al. Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nat Chem Biol 2015;11(1):64-70 Bozzi A, Saliola M, Falcone C, et al. Structural and biochemical studies of alcohol dehydrogenase isozymes from Kluyveromyces lactis. Biochim Biophys Acta 1997;1339(1):133-42 Bucciarelli T, Saliola M, Brisdelli F, et al. Oxidation of Cys278 of ADH I isozyme from kluyveromyces lactis by naturally occurring disulfides causes its reversible inactivation. Biochim Biophys Acta 2009; 1794(3):563-8 Sommer A, Traut RR. Diagonal polyacrylamide-dodecyl sulfate gel electrophoresis for the identification of ribosomal proteins crosslinked with methyl-4-mercaptobutyrimidate. Proc Natl Acad Sci USA 1974;71(10):3946-50

doi: 10.1586/14789450.2015.1069189

74.

Brennan JP, Wait R, Begum S, et al. Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J Biol Chem 2004;279(40):41352-60

..

Together with 73, these papers comprehensively describe the protocol of diagonal gel electrophoresis for mapping cellular thiol state.

75.

Mata-Cabana A, Florencio FJ, Lindahl M. Membrane proteins from the cyanobacterium synechocystis sp. PCC 6803 interacting with thioredoxin. Proteomics 2007;7(21):3953-63

76.

Wait R, Begum S, Brambilla D, et al. Redox options in two-dimensional electrophoresis. Amino Acids 2005;28(3): 239-72

77.

Jurado J, Fuentes-Almagro CA, Guardiola FA, et al. Proteomic profile of the skin mucus of farmed gilthead seabream (Sparus aurata). J Proteomics 2015;120: 21-34

78.

Oliveira BM, Coorssen JR, Martins-de-Souza D. 2DE: the phoenix of proteomics. J Proteomics 2014;104:140-50

79.

Thiede B, Koehler CJ, Strozynski M, et al. High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer. Mol Cell Proteomics 2013;12(2):529-38

80.

81.

82.

Izquierdo-Alvarez A, Ramos E, Villanueva J, et al. Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia. J Proteomics 2012;75(17):5449-62 Viswanathan S, Unlu M, Minden JS. Two-dimensional difference gel electrophoresis. Nat Protoc 2006;1(3): 1351-8 Hurd TR, Prime TA, Harbour ME, et al. Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling. J Biol Chem 2007;282(30):22040-51

83.

Osterman IA, Ustinov AV, Evdokimov DV, et al. A nascent proteome study combining click chemistry with 2DE. Proteomics 2013; 13(1):17-21

84.

Wu CL, Chou HC, Cheng CS, et al. Proteomic analysis of UVB-induced protein expression- and redox-dependent changes in skin fibroblasts using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis. J Proteomics 2012;75(7): 1991-2014

85.

Poschmann G, Grzendowski M, Stefanski A, et al. Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress. Biochim Biophys Acta 2015;1854(6):624-31

86.

Fan CY, Chou HC, Lo YW, et al. Proteomic and redox-proteomic study on the role of glutathione reductase in human lung cancer cells. Electrophoresis 2013; 34(24):3305-14

87.

Chou HC, Lu YC, Cheng CS, et al. Proteomic and redox-proteomic analysis of berberine-induced cytotoxicity in breast cancer cells. J proteomics 2012;75(11): 3158-76

88.

Seo YH, Carroll KS. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies. Proc Natl Acad Sci USA 2009;106(38):16163-8

89.

Peralta D, Bronowska AK, Morgan B, et al. A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation. Nat Chem Biol 2015;11(2): 156-63

90.

Maller C, Schroder E, Eaton P. Glyceraldehyde 3-phosphate dehydrogenase is unlikely to mediate hydrogen peroxide signaling: studies with a novel anti-dimedone sulfenic acid antibody. Anti redox signal 2011;14(1):49-60

91.

Signorini C, Leoncini S, De Felice C, et al. Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome. Oxid Med Cell Longev 2014;2014:195935

92.

Holmstrom KM, Finkel T. Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol 2014;15(6):411-21

93.

Hansen RE, Roth D, Winther JR. Quantifying the global cellular thiol-disulfide status. Proc Natl Acad Sci USA 2009;106(2):422-7

..

Together with 33, these papers comprehensively describe the pros and cons of various thiol-trapping strategies.



94.


95.

96.

97.

98.

Held JM, Danielson SR, Behring JB, et al. Targeted quantitation of site-specific cysteine oxidation in endogenous proteins using a differential alkylation and multiple reaction monitoring mass spectrometry approach. Mol Cell Proteomics 2010;9(7): 1400-10 Paulech J, Liddy KA, Engholm-Keller K, et al. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic Acid post-translational modifications. Mol Cell Proteomics 2015; 14(3):609-20 Martinez-Acedo P, Nunez E, Gomez FJ, et al. A novel strategy for global analysis of the dynamic thiol redox proteome. Mol Cell Proteomics 2012;11(9):800-13 Chi BK, Gronau K, Mader U, et al. S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics. Mol Cell Proteomics 2011; 10(11):M111 009506 Wang K, Huang C, Nice E. Recent advances in proteomics: towards the human proteome. Biomed Chromatogr 2014;28(6): 848-57

99.

Zhang Y, Cui W, Zhang H, et al. Electrochemistry-assisted top-down characterization of disulfide-containing proteins. Anal Chem 2012;84(8):3838-42

100.

Qu Z, Meng F, Zhou H, et al. NitroDIGE analysis reveals inhibition of protein S-nitrosylation by epigallocatechin gallates in lipopolysaccharide-stimulated microglial cells. J Neuroinflammation 2014;11:17

101.

102.

103.

Garcia-Santamarina S, Boronat S, Espadas G, et al. The oxidized thiol proteome in fission yeast–optimization of an ICAT-based method to identify H2O2-oxidized proteins. J proteomics 2011;74(11):2476-86 Sethuraman M, McComb ME, Huang H, et al. Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures. J Proteome Res 2004;3(6):1228-33 Kim SS, Rhee S, Lee KH, et al. Inhibitors of the proteasome block the myogenic differentiation of rat L6 myoblasts. FEBS lett 1998;433(1-2):47-50


104.

..

105.

106.

107.

Review

Garcia-Santamarina S, Boronat S, Domenech A, et al. Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry. Nat Protoc 2014;9(5):1131-45

114.

Together with 110, these papers comprehensively present protocols of OxICAT and ICAT-based method for detection redox state of proteins.

Chiappetta G, Ndiaye S, Igbaria A, et al. Proteome screens for cys residues oxidation: the redoxome. Methods Enzymol 2010;473: 199-216

115.

Liu P, Zhang H, Wang H, Xia Y. identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method. Proteomics 2014;14(6):750-62

.

This paper provides a novel approach termed OxiTRAQ which use a modified biotinswitch method to discern thiol-labeling and purification.

116.

McDonagh B, Martinez-Acedo P, Vazquez J , et al. Application of iTRAQ reagents to relatively quantify the reversible redox state of cysteine residues. Int J Proteomics 2012;2012:514847

117.

Thompson A, Schafer J, Kuhn K, et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 2003;75(8):1895-904

Christoforou AL, Lilley KS. Isobaric tagging approaches in quantitative proteomics: the ups and downs. Anal Bioanal Chem 2012; 404(4):1029-37 Maity S, Basak T, Bhat A, et al. Cross-compartment proteostasis regulation during redox imbalance induced ER stress. Proteomics 2014;14(15):1724-36 Wojdyla K, Williamson J, Roepstorff P, Rogowska-Wrzesinska A. The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations. J Proteomics 2015;113:415-34

elegans. Antioxid Redox Signal 2011;14(6): 1023-37

108.

Parker J, Balmant K, Zhu F, et al. cysTMTRAQ-An integrative method for unbiased thiol-based redox proteomics. Mol Cell Proteomics 2015;14(1):237-42

118.

109.

McDonagh B, Sakellariou GK, Smith NT, et al. Differential cysteine labeling and global label-free proteomics reveals an altered metabolic state in skeletal muscle aging. J Proteome Res 2014;13(11):5008-21

Pierce A, Unwin RD, Evans CA, et al. Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases. Mol Cell Proteomics 2008;7(5): 853-63

119.

110.

Leichert LI, Gehrke F, Gudiseva HV, et al. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. Proc Natl Acad Sci USA 2008;105(24):8197-202

Yang B, Wu YJ, Zhu M, et al. Identification of cross-linked peptides from complex samples. Nat Methods 2012;9(9): 904-6

120.

..

Together with 104, these papers comprehensively present protocols of OxICAT and ICAT-based method for detection redox state of proteins.

Lu S, Fan SB, Yang B, et al. Mapping native disulfide bonds at a proteome scale. Nat Methods 2015;12(4):329-31

121.

Depuydt M, Messens J, Collet JF. How proteins form disulfide bonds. Anti redox signal 2011;15(1):49-66

122.

Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11(3):298-300

123.

Neish AS, Gewirtz AT, Zeng H, et al. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 2000;289(5484): 1560-3

111.

Baker MA, Weinberg A, Hetherington L, et al. Analysis of protein thiol changes occurring during rat sperm epididymal maturation. Biol Reprod 2015;92(1):11

112.

Knoefler D, Thamsen M, Koniczek M, et al. Quantitative in vivo redox sensors uncover oxidative stress as an early event in life. Mol Cell 2012;47(5):767-76

113.

Kumsta C, Thamsen M, Jakob U. Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of caenorhabditis

doi: 10.1586/14789450.2015.1069189

Dissecting Redox Biology Using Fluorescent Protein Sensors.

Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method.

Cellular Redox Profiling Using High-content Microscopy.

Illuminating redox biology using NADH- and NADPH-specific sensors.

Preface: gas and redox sensors special issue.

Differential alkylation-based redox proteomics--Lessons learnt.

Thiol-based redox proteomics in cancer research.

ßNp63 controls cellular redox status.

biomedical promises.

Redox Proteomics and Platelet Activation: Understanding the Redox Proteome to Improve Platelet Quality for Transfusion.

ROSics: chemistry and proteomics of cysteine modifications in redox biology.

Hydrogen Sulfide and Cellular Redox Homeostasis.

The cellular redox environment alters antigen presentation.

cysTMTRAQ-An integrative method for unbiased thiol-based redox proteomics.

Extracellular Cysteine in Connexins: Role as Redox Sensors.

Mass spectrometry and redox proteomics: applications in disease.

Analytical approaches to the diagnosis and treatment of aging and aging-related disease: redox status and proteomics.

Modulation of nuclear receptor function by cellular redox poise.

Cellular mechanisms and physiological consequences of redox-dependent signalling.

The Tumorigenic Roles of the Cellular REDOX Regulatory Systems.

Modulation of cellular redox homeostasis by the endocannabinoid system.

Assessment of Cellular Redox State Using NAD(P)H Fluorescence Intensity and Lifetime.

Oxidative stress, redox regulation and diseases of cellular differentiation.

Cellular polarity in aging: role of redox regulation and nutrition.