ReviewProteomic methods for analysis of S-nitrosation☆
Introduction
Of all the potential post-translational thiol modifications that have been suggested to be involved in the transmission of intracellular signals, S-nitrosation is perhaps the most highly cited [1]. S-Nitrosation involves the modification of a thiol (RSH) to an S-nitrosothiol (RSNO). This can occur on low molecular weight or protein thiols to form a low molecular weight RSNO (lmwtRSNO) or a protein RSNO (pRSNO), respectively. This is thought to occur as a result of biological nitric oxide (NO) formation and has been thought of as a mechanism by which NO can transmit signals both within and between cells and tissues. While a great deal of information regarding S-nitrosation as a signaling paradigm has been obtained, there are still many unanswered questions. The idea that NO itself can trigger an intracellular signal through its interaction with thiols is at first a rather unlikely one. Metal centers (e.g. ferrous hemes) and free radicals (e.g. superoxide) are kinetically preferable targets, and NO does not react with thiols at any biologically meaningful rate [2], [3]. The direct reaction between NO and thiols is a redox reaction generating nitrous oxide, sulfenic acid and/or disulfide, but not RSNO [4], [5]. However, in the presence of oxygen and thiols, NO generates colored RSNO. These compounds were shown to have some synthetic chemical use, for instance as intermediates in the formation of mixed disulfides [6], and had antibacterial properties [7]. It was not until S-nitroso-N-acetyl-penicillamine (SNAP) was shown to have vasodilatory properties [8] that the biological potential of RSNO in mammalian systems became apparent. With the discovery of NO as a physiological mediator of vascular responses [9], [10], as well as many other processes, the role of endogenously produced RSNO, first measured by Stamler et al. [11], as mediators of a sub-set of NO-dependent responses became, and remains, an area of great interest.
While in vivo mechanisms of S-nitrosation remain the subject of debate, and are clearly more complex than a simple association of NO with a thiol, RSNO can be detected in vivo under basal and pathological conditions [11], [12], [13], [14]. Much investigation has focused on examining the role of S-nitrosation in modifying specific cellular pathways. For example, the ability of NO to affect apoptosis has been linked to modification of a catalytically important thiol in caspase-3 [15], [16]. Relatively few studies have examined S-nitrosation from a more global perspective by placing individual effects of nitrosative stress in the context of the proteome of S-nitrosated (or otherwise modified) thiols. This article will assess current methods for the detection of global protein S-nitrosation using proteomic techniques.
Section snippets
The Chemistry of S-Nitrosothiols
As the mechanism of thiol nitrosation is important in attempting to understand the S-nitrosated proteome, in this section we will briefly describe mechanisms of S-nitrosothiol formation. There are four major mechanisms of S-nitrosation that have the potential to occur in biological systems. (i) S-Nitrosothiols can be formed from the reaction of nitrous acid (HONO) with thiols (Eqs. (1) and (2)).HONO + H+ → H2ONO+H2ONO+ + RSH → RSNO+H2O + H+This is major mechanism of RSNO synthesis in the test tube [17],
What is Meant by “the Proteome of S-Nitrosated Proteins”?
This sounds like a simple question but one that has no simple answer. With currently available methods, it is fair to say that the levels of protein S-nitrosothiols generated in vivo are too low to be subject to proteomic analysis in any meaningful way [14]. Although some studies have reportedly examined the proteome of S-nitrosation from endogenous NO synthase sources [42], [43], [44], most have relied on exogenous treatments with NO or related compounds to increase the total intracellular
The Use of lmwtRSNO Transport to Titrate the S-Nitrosated Proteome
Although lmwtRSNO are often used in cell culture as NO donors, several lines of evidence suggest that the interactions between these compounds and cells are much more complex than simple NO release. Firstly, RSNO are relatively stable in metal ion-free buffers in the dark, and in cell culture their decomposition is driven by the presence of cells [55]. We have demonstrated that cystine, present in most cell culture medium, is essential for cell-mediated GSNO metabolism [56]. Secondly, there are
Challenges for the Detection of the S-Nitroso Functional Group
It is relatively simple to detect S-nitrosothiols at levels over ∼100 pmol/mg protein using either spectrophotometric, fluorimetric or chemiluminescence methods. In our hands, tri-iodide-based chemiluminescence [63], [64] can accurately detect levels of S-nitrosothiol in cell lysate down to 1–2 pmol/mg protein [65]. However, these methods only detect the total S-nitrosothiol content and do not identify which proteins are modified.
Traditional methods for the determination of protein modifications
Conclusion
In this review we have detailed methodologies to identify the S-nitrosated proteome. It is difficult as yet to conclude which strategy will be the most useful, but direct peptide-capture methods have provided the most compelling picture of the S-nitrosated proteome in publications to date. In all of the methods described, one has to be aware that the detection methodology is indirect and relies on chemical modification of the S-nitroso group. Consequently, the specificity of this modification
Acknowledgements
This work was supported by NIH grant GM55792.
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This paper is part of a special issue entitled “Analysis of the l-Arginine/NO pathway”, guest edited by D. Tsikas.