Proteomics 2014, 00, 1–13
1
DOI 10.1002/pmic.201400164
REVIEW
Thiol-based redox proteomics in cancer research Kefei Yuan1∗∗ , Yuan Liu1∗∗ , Hai-Ning Chen2∗∗ , Lu Zhang1 , Jiang Lan1 , Wei Gao1 , Qianhui Dou1 , Edouard C. Nice3,4∗ and Canhua Huang1 1
The State Key Laboratory for Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R. China Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, P. R. China 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia 4 The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R. China 2
Cancer cells maintain their intracellular ROS concentrations at required levels for their survival. Changes in ROS concentrations can regulate biochemical signaling mechanisms that control cell function. It has been demonstrated that ROS regulate the cellular events through redox regulation of redox-sensitive proteins (redox sensors). Upon oxidative stress, redox sensors undergo redox modifications that cause the allosteric changes of these proteins and endow them with different functions. Understanding the altered functions of redox sensors and the underlying mechanisms is critical for the development of novel cancer therapeutics. Recently, a series of high-throughput proteomics approaches have been developed for screening redox processes. In this manuscript, we review these methodologies and discuss the important redox sensors recently identified that are related to cancer.
Received: April 25, 2014 Revised: August 15, 2014 Accepted: September 18, 2014
Keywords: Biomedicine / Cancer biomarkers / Comparative proteomics / Mass spectrometry / Qxidative stress / Signal transduction
1
Introduction
Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries [1]. The global burden of cancer continues to increase largely because of the aging and expanding world population coupled with increasing adoption of cancercausing behaviors, particularly diet and smoking. Based on GLOBOCAN estimates, about 14.1 million cancer cases and
Correspondence: Professor Canhua Huang, The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, P. R. China E-mail: [email protected] Fax: +86-28-85164060 Abbreviations: AMPK, 5 -AMP-activated protein kinase; ASK1, apoptosis signal-regulating kinase 1; ATM, ataxia telangiectasia mutated; cGMP, guanosine 3 ,5 -monophosphate; CHIP, carboxyl terminus of Hsc70-interacting protein; FasL, Fas ligand; GPx, glutathione peroxidase; HIPK2, homeodomain-interacting protein kinase 2; HMGB1, High-mobility group box 1 protein; IAM, iodoacetamide; isoTOP-ABPP, isotopic tandem orthogonal proteolysis activity based protein profiling; MAP, mitogen-activated protein; PKM2, Pyruvate kinase M2; SENPs, Sentrin/SUMO-specific proteases; TMT, tandem mass tag; TNF-␣, tumor necrosis factor-␣; Trx, thioredoxin; VEGF, vascular endothelial growth factor
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
8.2 million cancer deaths are estimated to have occurred in 2012 [2]. Although methods for cancer diagnosis and therapeutic intervention have been significantly improved over the years, many critical questions, which markedly impair the effect of current clinical cancer treatments, remain unanswered, including the development of drug resistance, as well as recurrence and metastasis. In order to overcome these problems, basic research is required to understand the mechanisms underlying them. ROS are formed and degraded by all aerobic organisms and have been demonstrated to play essential roles in a broad range of physiological and pathophysiological processes, such as signaling [3, 4], regulation of vascular tone [5, 6], tissue injury [7, 8], and control of inflammation [9, 10]. Accumulating evidence has demonstrated that increased generation of ROS and an altered redox status can be observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells is closely related to the development and progression of cancer [11]. On the other hand, many signaling pathways that are linked to tumorigenesis can also regulate the metabolism of ROS through direct or indirect mechanisms ∗ Additional corresponding author: Professor Edouard C. Nice, E-mail: [email protected] ∗∗ These authors contributed equally to this work. Colour Online: See the article online to view Figs. 1–3 in colour.
www.proteomics-journal.com
2
K. Yuan et al.
[12]. The effects of ROS on cellular biological events are both dose and context dependent. High ROS levels are generally detrimental to cells, including cancer cells [13]. However, ROS can also promote tumor formation by inducing DNA mutations and pro-oncogenic signaling pathways [14]. Proteins are the major targets of oxidants and can undergo reversible redox reactions because of their high overall abundance in biological systems. Although the side chain of almost every amino acid can be damaged by oxidative stress, the thiol group of the amino acid cysteine has been proved to be the primary target for oxidation reactions. Redox reversible protein modifications (redox regulation), particularly thiol oxidation, are recognized to be an early cellular response to oxidative stress, and may also play an important role in redox signaling pathways [15]. Recent studies revealed that ROS-mediated redox regulation of proteins is involved in the pathogenesis and/or progression of many human diseases, including cancer, suggesting that in order to understand the contradictory effects of ROS, it is crucial to analyze the detailed redox status of key proteins that are implicated in tumorigenesis [16]. Many powerful approaches have been developed to examine the redox regulation of proteins. Among them, redox proteomics has been recognized as being ideally suited for the identification of ROS-induced protein modifications both in redox signaling and under oxidative stress conditions. Using different redox proteomics approaches, many studies have identified specific redox modifications of proteins in various human diseases, such as cancer [16, 17], neurodegenerative disorders [18–20], cardiovascular disease [21], as well as alkaptonuria [22] and sickle cell disease [23]. Besides cancer, neurodegenerative disorders have also been a focus of redox proteomics studies. Novel proteomic methods have been used to interrogate the redox-regulated mechanisms involved in the development of Alzheimer disease [18,20], Parkinson disease [24], as well as aging [19]. These studies have provided mechanistic information on the development of the diseases and insights into the pathways involved in their pathogenesis as well as into downstream functional consequences [25]. In this review, we will focus on the thiol-based redox regulation of proteins that are related to cancer, which is a major disease burden worldwide: each year, tens of millions of people are diagnosed with the disease and more than half of the patients eventually die from it. We will briefly introduce several novel effective redox proteomics approaches. Then, we will summarize the most important discoveries in the study of redox processes related to the development and progression of cancer, illustrating the underlying thiol modifications that may serve as the blueprint for developing novel treatments against cancer [26].
2
Redox regulation screening by redox proteomics
Currently, two major approaches, gel-based and nongel methods, are used in redox proteomics research. With recent C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2014, 00, 1–13
developments in MS, such as iTRAQ [27], SRM (which is also termed MRM) [28], as well as stable isotope standards and capture by antipeptide antibodies (SISCAPA) [29], the nongel proteomic approach has become the method of choice because of its efficiency and convenience. Compared to the traditional gel-based method that is more complicated, the nongel proteomic approach requires only three steps: digestion and labeling of resultant peptides, the separation of peptides, and automated data acquisition. Below, we discuss four key examples of these recently reported nongel proteomic approaches, which hold the potential for identification of novel redox regulations associated with cancer (Fig. 1). 2.1 OxICAT The OxICAT method was developed by combining a thioltrapping technique with the established ICAT technology [30]. It uses the thiol-trapping reagent iodoacetamide (IAM) to label the accessible cysteines in proteins in a rapid and irreversible manner [31]. The method requires the ICAT reagents, which are a cleavable biotin affinity tag, the IAM moiety, and a 9-carbon linker, which includes an isotopically light 12 Clinker (the light ICAT reagent) and a 9-Da heavier isotopically heavy 13 C-linker (the heavy ICAT reagent) [31]. The OxICAT procedure starts with labeling of reduced cysteines. The samples are lysed by denaturing buffer to expose all the reduced cysteines of proteins. Then the light ICAT reagents are added to the lysate to label the reduced cysteines irreversibly. The lysate is then treated with a strong thiol reductant Tris(2carboxyethyl)phosphine to reduce all the reversible oxidative thiol modifications, and the newly accessible cysteines are labeled with the heavy ICAT reagent. The samples are then digested with trypsin and purified via the biotin tag. Finally, all the purified ICAT-labeled peptides are identified and quantified by LC-MS/MS [31]. An open-source program msInspect is used to analyze the MS data. [32]. Mass signals, which share the same elution profiles yet have a mass difference of 9 Da or multiples (9 Da additional mass per heavy ICAT) are separated from the MS data to generate “ICAT pairs” [31]. In this manner, the OxICAT method can quantify the reduced and oxidized forms of certain proteins, which have accessible cysteines labeled with IAM [31]. 2.2 cysTMT The tandem mass tag (TMT) approach, which enables concurrent identification and multiplexed quantitation of proteins in different samples, is a well-established multiplex MS analysis method. CysTMT is a version of the TMT approach that is thiol reactive [33].The cysTMT reagents, which are several isobaric (mass and structure) isomers, can be used to label the sulfhydryl (-SH) groups irreversibly. Similar to the reagents used in the OxICAT method, cysTMT reagents react specifically with reduced cysteines in peptides and proteins. After labeling, peptides with various cysteine modifications, www.proteomics-journal.com
Proteomics 2014, 00, 1–13
3
Figure 1. Nongel redox proteomic approaches. For OxICAT, the reduced proteins are irreversibly labeled with 12CICAT first. Then, the oxidized proteins are reduced by Tris(2-carboxyethyl)phosphine (TCEP) and labeled with 13C-ICAT. After that, the proteome is digested and purified for further analysis with LC-MS/MS. For cysTMT, the reduced proteins are irreversibly labeled with cysTMT 126 first. Then, the oxidized proteins are reduced by TCEP and labeled with other cysTMT tags (from 127 to 131). After that, the proteome is digested and purified for further analysis with LC-MS/MS. For isoTOP-ABPP, the reduced proteins of both experimental and control group are labeled with IA probe first. Then, the IA-labeled proteins of the experimental group are further labeled with light tobacco-etch virus (TEV) tag, while the IA-labeled proteins of the control group are further labeled with heavy TEV tag. After that, the proteome is enriched and digested for further analysis with LC-MS/MS. For q-oxPTPome, the reduced proteins are irreversibly labeled with N-ethylmaleimide first. Then, the oxidized proteins are reduced by DTT and then oxidized to sulfonic acid by pervanadate (PV). After that, the proteome is digested and purified for further analysis with LC-MS/MS.
such as oxidation, disulfide bonds, and S-nitrosylation can be identified and quantified by MS. The Libra module of the transproteome pipeline is used for collecting the quantitative values of the TMT reporter ions [34]. Additionally, the exact position of each cysTMT6 -modified cysteine can be determined by mapping the peptide against the full protein sequence. For data analysis, reporter ions are normalized to total reporter ion signal within each spectrum and then values are averaged across all peptide observations for an individual site. Compared with the traditional biotin switch technique that has been widely used for identification of protein S-nitrosylation [35], this new reagent fulfills the requirements for a biotin switch label and offers some distinct advantages, including a permanent mass tag and the fragmentation of up to six isotopically balanced reporter ions between 126 and 131 Da permitting multiplex quantification [36]. 2.3 isoTOP-ABPP Originally, the isotopic tandem orthogonal proteolysis activity based protein profiling (isoTOP-ABPP) approach was recognized as a tool for quantifying the active cysteine residues in proteomes via a clickable IAM alkyne probe [37]. A recent study has introduced this method for the identification
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and quantification of reactive cysteines in native proteomes [37]. The competitive profiling approach has many advantages, including its sensitivity to different forms of cysteine oxidation (i.e., it is sensitive to all the oxidative modifications that impair the nucleophilicity of the IA probe reactive cysteines) [38]. In the first step of isoTOP-ABPP, cell extracts are treated with the IA probe to alkylate all reactive cysteines in the proteome and protect them from further oxidative modifications. Then, the copper-catalyzed click chemistry [39, 40] is used to attach the protease-cleavable azido-biotin tag [37] to the alkyne. Next, isotopic light (12 C and 14 N) and heavy (13 C and 15 N) tags are conjugated to the control and experimental group samples, respectively. After that, equal amounts of the control and experimental group samples are combined and the mixture is subjected to successive protease digestion by trypsin and tobacco-etch virus protease to yield cysteinecontaining peptides with isotopic tags for MS analysis. The ratio of light/heavy form represents the relative reactivity of cysteines with the IA probe in the proteome of the control group versus the experimental group. If a cysteine is oxidized, its reactivity with the IA probe decreases and the light/heavy ratio is
1
DOI 10.1002/pmic.201400164
REVIEW
Thiol-based redox proteomics in cancer research Kefei Yuan1∗∗ , Yuan Liu1∗∗ , Hai-Ning Chen2∗∗ , Lu Zhang1 , Jiang Lan1 , Wei Gao1 , Qianhui Dou1 , Edouard C. Nice3,4∗ and Canhua Huang1 1
The State Key Laboratory for Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R. China Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, Chengdu, P. R. China 3 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia 4 The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, P. R. China 2
Cancer cells maintain their intracellular ROS concentrations at required levels for their survival. Changes in ROS concentrations can regulate biochemical signaling mechanisms that control cell function. It has been demonstrated that ROS regulate the cellular events through redox regulation of redox-sensitive proteins (redox sensors). Upon oxidative stress, redox sensors undergo redox modifications that cause the allosteric changes of these proteins and endow them with different functions. Understanding the altered functions of redox sensors and the underlying mechanisms is critical for the development of novel cancer therapeutics. Recently, a series of high-throughput proteomics approaches have been developed for screening redox processes. In this manuscript, we review these methodologies and discuss the important redox sensors recently identified that are related to cancer.
Received: April 25, 2014 Revised: August 15, 2014 Accepted: September 18, 2014
Keywords: Biomedicine / Cancer biomarkers / Comparative proteomics / Mass spectrometry / Qxidative stress / Signal transduction
1
Introduction
Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries [1]. The global burden of cancer continues to increase largely because of the aging and expanding world population coupled with increasing adoption of cancercausing behaviors, particularly diet and smoking. Based on GLOBOCAN estimates, about 14.1 million cancer cases and
Correspondence: Professor Canhua Huang, The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, P. R. China E-mail: [email protected] Fax: +86-28-85164060 Abbreviations: AMPK, 5 -AMP-activated protein kinase; ASK1, apoptosis signal-regulating kinase 1; ATM, ataxia telangiectasia mutated; cGMP, guanosine 3 ,5 -monophosphate; CHIP, carboxyl terminus of Hsc70-interacting protein; FasL, Fas ligand; GPx, glutathione peroxidase; HIPK2, homeodomain-interacting protein kinase 2; HMGB1, High-mobility group box 1 protein; IAM, iodoacetamide; isoTOP-ABPP, isotopic tandem orthogonal proteolysis activity based protein profiling; MAP, mitogen-activated protein; PKM2, Pyruvate kinase M2; SENPs, Sentrin/SUMO-specific proteases; TMT, tandem mass tag; TNF-␣, tumor necrosis factor-␣; Trx, thioredoxin; VEGF, vascular endothelial growth factor
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
8.2 million cancer deaths are estimated to have occurred in 2012 [2]. Although methods for cancer diagnosis and therapeutic intervention have been significantly improved over the years, many critical questions, which markedly impair the effect of current clinical cancer treatments, remain unanswered, including the development of drug resistance, as well as recurrence and metastasis. In order to overcome these problems, basic research is required to understand the mechanisms underlying them. ROS are formed and degraded by all aerobic organisms and have been demonstrated to play essential roles in a broad range of physiological and pathophysiological processes, such as signaling [3, 4], regulation of vascular tone [5, 6], tissue injury [7, 8], and control of inflammation [9, 10]. Accumulating evidence has demonstrated that increased generation of ROS and an altered redox status can be observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells is closely related to the development and progression of cancer [11]. On the other hand, many signaling pathways that are linked to tumorigenesis can also regulate the metabolism of ROS through direct or indirect mechanisms ∗ Additional corresponding author: Professor Edouard C. Nice, E-mail: [email protected] ∗∗ These authors contributed equally to this work. Colour Online: See the article online to view Figs. 1–3 in colour.
www.proteomics-journal.com
2
K. Yuan et al.
[12]. The effects of ROS on cellular biological events are both dose and context dependent. High ROS levels are generally detrimental to cells, including cancer cells [13]. However, ROS can also promote tumor formation by inducing DNA mutations and pro-oncogenic signaling pathways [14]. Proteins are the major targets of oxidants and can undergo reversible redox reactions because of their high overall abundance in biological systems. Although the side chain of almost every amino acid can be damaged by oxidative stress, the thiol group of the amino acid cysteine has been proved to be the primary target for oxidation reactions. Redox reversible protein modifications (redox regulation), particularly thiol oxidation, are recognized to be an early cellular response to oxidative stress, and may also play an important role in redox signaling pathways [15]. Recent studies revealed that ROS-mediated redox regulation of proteins is involved in the pathogenesis and/or progression of many human diseases, including cancer, suggesting that in order to understand the contradictory effects of ROS, it is crucial to analyze the detailed redox status of key proteins that are implicated in tumorigenesis [16]. Many powerful approaches have been developed to examine the redox regulation of proteins. Among them, redox proteomics has been recognized as being ideally suited for the identification of ROS-induced protein modifications both in redox signaling and under oxidative stress conditions. Using different redox proteomics approaches, many studies have identified specific redox modifications of proteins in various human diseases, such as cancer [16, 17], neurodegenerative disorders [18–20], cardiovascular disease [21], as well as alkaptonuria [22] and sickle cell disease [23]. Besides cancer, neurodegenerative disorders have also been a focus of redox proteomics studies. Novel proteomic methods have been used to interrogate the redox-regulated mechanisms involved in the development of Alzheimer disease [18,20], Parkinson disease [24], as well as aging [19]. These studies have provided mechanistic information on the development of the diseases and insights into the pathways involved in their pathogenesis as well as into downstream functional consequences [25]. In this review, we will focus on the thiol-based redox regulation of proteins that are related to cancer, which is a major disease burden worldwide: each year, tens of millions of people are diagnosed with the disease and more than half of the patients eventually die from it. We will briefly introduce several novel effective redox proteomics approaches. Then, we will summarize the most important discoveries in the study of redox processes related to the development and progression of cancer, illustrating the underlying thiol modifications that may serve as the blueprint for developing novel treatments against cancer [26].
2
Redox regulation screening by redox proteomics
Currently, two major approaches, gel-based and nongel methods, are used in redox proteomics research. With recent C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2014, 00, 1–13
developments in MS, such as iTRAQ [27], SRM (which is also termed MRM) [28], as well as stable isotope standards and capture by antipeptide antibodies (SISCAPA) [29], the nongel proteomic approach has become the method of choice because of its efficiency and convenience. Compared to the traditional gel-based method that is more complicated, the nongel proteomic approach requires only three steps: digestion and labeling of resultant peptides, the separation of peptides, and automated data acquisition. Below, we discuss four key examples of these recently reported nongel proteomic approaches, which hold the potential for identification of novel redox regulations associated with cancer (Fig. 1). 2.1 OxICAT The OxICAT method was developed by combining a thioltrapping technique with the established ICAT technology [30]. It uses the thiol-trapping reagent iodoacetamide (IAM) to label the accessible cysteines in proteins in a rapid and irreversible manner [31]. The method requires the ICAT reagents, which are a cleavable biotin affinity tag, the IAM moiety, and a 9-carbon linker, which includes an isotopically light 12 Clinker (the light ICAT reagent) and a 9-Da heavier isotopically heavy 13 C-linker (the heavy ICAT reagent) [31]. The OxICAT procedure starts with labeling of reduced cysteines. The samples are lysed by denaturing buffer to expose all the reduced cysteines of proteins. Then the light ICAT reagents are added to the lysate to label the reduced cysteines irreversibly. The lysate is then treated with a strong thiol reductant Tris(2carboxyethyl)phosphine to reduce all the reversible oxidative thiol modifications, and the newly accessible cysteines are labeled with the heavy ICAT reagent. The samples are then digested with trypsin and purified via the biotin tag. Finally, all the purified ICAT-labeled peptides are identified and quantified by LC-MS/MS [31]. An open-source program msInspect is used to analyze the MS data. [32]. Mass signals, which share the same elution profiles yet have a mass difference of 9 Da or multiples (9 Da additional mass per heavy ICAT) are separated from the MS data to generate “ICAT pairs” [31]. In this manner, the OxICAT method can quantify the reduced and oxidized forms of certain proteins, which have accessible cysteines labeled with IAM [31]. 2.2 cysTMT The tandem mass tag (TMT) approach, which enables concurrent identification and multiplexed quantitation of proteins in different samples, is a well-established multiplex MS analysis method. CysTMT is a version of the TMT approach that is thiol reactive [33].The cysTMT reagents, which are several isobaric (mass and structure) isomers, can be used to label the sulfhydryl (-SH) groups irreversibly. Similar to the reagents used in the OxICAT method, cysTMT reagents react specifically with reduced cysteines in peptides and proteins. After labeling, peptides with various cysteine modifications, www.proteomics-journal.com
Proteomics 2014, 00, 1–13
3
Figure 1. Nongel redox proteomic approaches. For OxICAT, the reduced proteins are irreversibly labeled with 12CICAT first. Then, the oxidized proteins are reduced by Tris(2-carboxyethyl)phosphine (TCEP) and labeled with 13C-ICAT. After that, the proteome is digested and purified for further analysis with LC-MS/MS. For cysTMT, the reduced proteins are irreversibly labeled with cysTMT 126 first. Then, the oxidized proteins are reduced by TCEP and labeled with other cysTMT tags (from 127 to 131). After that, the proteome is digested and purified for further analysis with LC-MS/MS. For isoTOP-ABPP, the reduced proteins of both experimental and control group are labeled with IA probe first. Then, the IA-labeled proteins of the experimental group are further labeled with light tobacco-etch virus (TEV) tag, while the IA-labeled proteins of the control group are further labeled with heavy TEV tag. After that, the proteome is enriched and digested for further analysis with LC-MS/MS. For q-oxPTPome, the reduced proteins are irreversibly labeled with N-ethylmaleimide first. Then, the oxidized proteins are reduced by DTT and then oxidized to sulfonic acid by pervanadate (PV). After that, the proteome is digested and purified for further analysis with LC-MS/MS.
such as oxidation, disulfide bonds, and S-nitrosylation can be identified and quantified by MS. The Libra module of the transproteome pipeline is used for collecting the quantitative values of the TMT reporter ions [34]. Additionally, the exact position of each cysTMT6 -modified cysteine can be determined by mapping the peptide against the full protein sequence. For data analysis, reporter ions are normalized to total reporter ion signal within each spectrum and then values are averaged across all peptide observations for an individual site. Compared with the traditional biotin switch technique that has been widely used for identification of protein S-nitrosylation [35], this new reagent fulfills the requirements for a biotin switch label and offers some distinct advantages, including a permanent mass tag and the fragmentation of up to six isotopically balanced reporter ions between 126 and 131 Da permitting multiplex quantification [36]. 2.3 isoTOP-ABPP Originally, the isotopic tandem orthogonal proteolysis activity based protein profiling (isoTOP-ABPP) approach was recognized as a tool for quantifying the active cysteine residues in proteomes via a clickable IAM alkyne probe [37]. A recent study has introduced this method for the identification
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and quantification of reactive cysteines in native proteomes [37]. The competitive profiling approach has many advantages, including its sensitivity to different forms of cysteine oxidation (i.e., it is sensitive to all the oxidative modifications that impair the nucleophilicity of the IA probe reactive cysteines) [38]. In the first step of isoTOP-ABPP, cell extracts are treated with the IA probe to alkylate all reactive cysteines in the proteome and protect them from further oxidative modifications. Then, the copper-catalyzed click chemistry [39, 40] is used to attach the protease-cleavable azido-biotin tag [37] to the alkyne. Next, isotopic light (12 C and 14 N) and heavy (13 C and 15 N) tags are conjugated to the control and experimental group samples, respectively. After that, equal amounts of the control and experimental group samples are combined and the mixture is subjected to successive protease digestion by trypsin and tobacco-etch virus protease to yield cysteinecontaining peptides with isotopic tags for MS analysis. The ratio of light/heavy form represents the relative reactivity of cysteines with the IA probe in the proteome of the control group versus the experimental group. If a cysteine is oxidized, its reactivity with the IA probe decreases and the light/heavy ratio is
