The SNO/SOH TMT strategy for combinatorial analysis of reversible cysteine oxidations

Highlights

• Proteome-wide detection and quantitation of cysteine S-nitrosylation and S-sulfenylation.

• Investigation of possible cross-talk/interaction between oxidised forms of cysteine.

• Correction of modification levels by protein abundance changes in a single nLC-MSMS experiment.

• Determination of relative modification site occupancy.

Abstract

Redox homeostasis is essential for normal function of cells and redox imbalance has been recognised as a pathogenic factor of numerous human diseases. Oxidative modifications of cysteine thiols modulate function of many proteins, mediate signalling, and fine-tune transcriptional and metabolic processes. In this study we present the SNO/SOH TMT strategy, which enables simultaneous analysis of two different types of cysteine modification: S-nitrosylation (SNO) and S-sulfenylation (SOH). The method facilitates quantitation of modification changes corrected by changes in protein abundance levels and estimation of relative modification site occupancy in a single nLC-MSMS run. The approach was evaluated in vivo using an Escherichia coli based model of mild oxidative stress. Bacteria were grown anaerobically on fumarate or nitrate. Short-term treatment with sub-millimolar levels of hydrogen peroxide was used to induce SOH. We have identified and quantified 114 SNO and SOH modified peptides. In many instances SNO and SOH occupy the same site, suggesting an association between them. High site occupancy does not equate to a site of modification which responds to redox imbalance. The SNO/SOH TMT strategy is a viable alternative to existing methods for cysteine oxidation analysis and provides new features that will facilitate our understanding of the interplay between SNO and SOH.

Biological significance

SNO/SOH TMT strategy outperforms other available strategies for cysteine oxidation analysis. It provides quantitative profiling of S-nitrosylation and S-sulfenylation changes simultaneously in two experimental conditions. It allows correction of modification levels by protein abundance changes and determination of relative modification site occupancy — all in a single nLC-MSMS experiment based on commercially available reagents. The method has proven precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale.

Introduction

Cellular thiol redox state is a crucial mediator of multiple metabolic, signalling, and transcriptional processes in cells. Balance between oxidising and reducing conditions is essential for the normal function and survival of cells. Oxidative stress has been recognised as pathogenic and etiological factor of numerous human diseases, including cancer, diabetes, atherosclerosis, cardiovascular and neurodegenerative diseases [1]. It is also involved in physiological ageing and degenerative processes that occur in age-related diseases. Protein thiols, due to their ability to be reversibly oxidised, are recognised as key components involved in the maintenance of redox homeostasis [2]. There are 7 recognised oxidative modifications of cysteine, 5 of which are reversible [2]. Among these, S-nitrosylation (SNO) and S-sulfenylation (SOH) play a significant role in regulating cellular signalling in response to redox imbalance. SNO is a covalent addition of nitroso group onto the reduced thiol of cysteine's side chain. Endogenous sources of nitroso group are for instance, the nitric oxide synthase (NOS) family of enzymes [3]. There are representatives of the NOS family in almost every cell type (neuronal nNOS/NOS1, endothelial eNOS/NOS3, inducible/Ca^2 +-independent iNOS/NOS2) [3]. Indirectly, SNO may also occur via contact with existing nitrosylated proteins that act as nitroso group donors, in a process termed trans-nitrosylation [4]. SOH is an oxidation of the cysteine sulfhydryl to sulfenic acid. The major inducer of SOH is hydrogen peroxide (H₂O₂), which is generated in the cell e.g., as a product of superoxide dismutase catalytic activity [5].

SNO and SOH do not occur equally over all cysteine residues due to the variable electronegativity of cysteine in biological systems. This, combined with their transient nature makes them excellent signalling mediators [1]. For example, in Escherichia coli grown anaerobically on nitrate, SNO of OxyR initiates a signalling cascade activating mechanisms protecting against endogenous nitrosative stress [6]. Antioxidant buffering capacity is also an important function of SNO. Elevated levels of SNO during ischemic preconditioning prevent build-up of irreversible protein oxidations helping to guard against irreversible oxidative damage during reperfusion [4]. SOH is known to function as a transient intermediate in the formation of more stable cysteine oxidation products [7] and it is an indicator of oxidant-sensitive cysteines and potentially also of severe oxidative damage [8]. It is implicated in redox regulation of transcription factors [9]. Protein SOH can modify the activity of enzymes [10], [11] and act as an initiator of disulfide bond formation [12].

For effective analysis of SNO and SOH selective labelling and enrichment are required [13]. A number of methods for analysis of cysteine oxidation exist, each with certain advantages and limitations, as reviewed in [2]. The majority of strategies used in the analysis of protein SNO, are variations of the biotin switch technique [14]. The most advanced of these provide precise localisation of modification site and its relative abundance [15], [16], [17]. Biotin switch has also been adapted for analysis of cysteine SOH [18]. However, dimedone labelling is more widespread [19], [20]. Recently, new cell-permeable analogues of dimedone have been developed which significantly shortens the time for sulfenic acid selective capture thus minimising artifactual results [21], [22].

Despite dynamic advances in molecular biology and mass spectrometry-based strategies for analysis of cysteine redox proteomes there remain several challenges. The majority of current methods provide quantitative information, yet, correction of modification levels by protein abundance changes is still a rare practise. In fact there are only few existing methods which either inherently correct observed modification levels in such a way [15], or with additional workload provide such correction [23], [24], [25]. However, all of these approaches use non-isobaric isotopic labels which suffer from low throughput due to increased sample complexity, limited dynamic range of quantitation and typically can target only 2 samples/conditions due to isotopic limitations. An additional layer of quantitative information relevant for determining the functional importance of a given modification is site occupancy [26]. Again, only rarely and at the expense of additional analytical steps has this been addressed [27], [28]. Finally, few methods exist that target multiple cysteine oxoforms. Attempts have been made to target the SNO and a pool of all reversibly oxidised cysteines in parallel [29]. However, further development of such strategies targeting individual modifications simultaneously is important to decode potential cross-talk between different cysteine oxoforms.

In addition to the limitations of existing methods the majority of studies investigating cysteine oxidative modifications use in vitro models where oxidation is induced chemically, for example by treatment with S-nitrosoglutathione (GSNO) [30], [31]. Such models do not reflect physiological levels of oxidative modifications. Additionally, they are often performed outside cellular environment and in denaturing conditions, which neglects the role of protein structure in the susceptibility of cysteine to oxidation.

Here we present an analytical strategy which provides quantitative analysis of cysteine SNO and SOH simultaneously including precise assignment of modification site. The method inherently corrects for protein abundance changes and permits determination of relative site occupancy of both modifications. To achieve this we make use of Iodoacetyl Tandem Mass Tags (iodoTMT™) which are a variant of the popular amine reactive TMT™ isobaric labels. Replacement of the amine reactive group with a thiol reactive iodoacetyl moiety allows for quantitative analysis of cysteine subproteomes by tandem mass spectrometry [32]. Enrichment or immunoassay based detection of iodoTMT™-containing peptides and proteins is achieved by the application of an anti-TMT™ antibody. As with their amine counterpart, isobaric iodoTMT™ tags allow for concurrent analysis of up to six samples [32]. In the presented configuration, our SNO/SOH TMT method allows analysis of two treatment groups in one experiment.

SNO/SOH TMT was developed and optimised to target near physiological levels of oxidative stress and to characterise proteins modified in native conditions. To test the applicability of the method to analysis of complex biological samples we have used SNO/SOH TMT strategy to analyse the thiol redox proteome of E. coli under mild oxidative stress induced in vivo. Our model was built upon previous works with minor alterations [6]. Induction of SNO was achieved by anaerobic growth in minimal media supplemented with nitrate, whereas fumarate was used in the control group. SOH was induced by brief in-culture stimulation with hydrogen peroxide (H₂O₂).

We show that the SNO/SOH TMT strategy provides robust and reproducible method for identification of the modification site and quantification of the relative abundance of SNO and SOH in proteins. Combined with anti-TMT™ enrichment it is sensitive enough to measure the endogenous levels of thiol modifications. Our results indicate that SNO and SOH coexist on several cysteine residues and only few of the modified residues change between low and mild oxidative stress conditions. The approach presented expands out tool-box for redox proteome analysis and opens up new possibilities for studying the interdependence of different cysteine oxoforms. We emphasise that although SNO/SOH TMT is a powerful methodology for screening of SNO and SOH alterations in response to changes in ROS/RNS levels, the information it provides should be evaluated further with alternative analytical strategies in order to reveal their true biological importance.