Brent R. Martin, Ph.D.
Department of Chemistry, University of Michigan

Cysteine oxidation by the gaseous second messenger nitric oxide leads to the reversible modification of select cysteine residues in proteins, termed either S-nitrosylation or S-nitrosation.¹ This modification is non-enzymatic and relies largely on a protein’s proximity to diffusible nitric oxide. Just as nitric oxide levels are tightly regulated by hormone-sensitive nitric oxide synthases, it follows that S-nitrosation can also be hormonally regulated. Indeed, inducible nitric oxide synthase stimulates the S-nitrosation of interacting proteins, which in turn exchange S-nitroso groups and transfer the modification to select sites on target proteins.² In hundreds of examples, S-nitrosation can modulate the function of cellular proteins, often by transient inactivation of functional cysteines in enzyme active sites. Classes of enzymes affected include metabolic enzymes, cysteine proteases, phosphatases, and ion channels.³ Since active site cysteines have lower pK_a values, they are more nucleophilic and more readily react with cellular electrophiles. Therefore, S-nitrosation and other oxidants favor functional cysteines, leading to direct modulation of cellular pathways.⁴ Solvent accessible S-nitrosated cysteines are readily exchanged with reduced glutathione, which then delivers S-nitrosoglutathione for further enzymatic reduction. In contrast, S-nitrosation also occurs on cysteines in protected protein environments, such as in the active site of enzymes. In this hindered environment, S-nitrosation can be long-lived and functionally block protein function to disrupt cellular pathways.

Given the broad role of S-nitrosation in cellular regulation, it is important to have robust, selective tools for biochemical analysis.⁵ Interest in this research area has fueled the development of chemical enrichment strategies to purify and analyze protein S-nitrosation, including ascorbate-dependent switch techniques, organomercury enrichment, phosphine ligations, and sulfinic acid catalyzed thiosulfonate formation.^6-13 All of these methods require complete alkylation of cellular thiols to prevent disulfide scrambling and exchange of the probe-linked adducts.

Biotin Switch Technique (BST)

The primary method to biochemically characterize protein S-nitrosation is termed the biotin switch technique or BST (Figure 1).^6,7 In this approach, cell lysates are first denatured in a buffered detergent solution supplemented with the metal chelator neocuproine, which prevents copper-catalyzed breakdown of S-nitrosothiols. S-Nitrosothiols are particularly light sensitive, so it is critical to avoid exposure to daylight to prevent photolysis and disulfide scrambling. After denaturation, the sample is treated with the cysteine alkylation agent methyl methane thiosulfonate (MMTS), which reacts with free thiols to form inert disulfides, releasing methylsulfinic acid. Next, the sample is incubated with ascorbate, which selectively reduces nitrosothiols to thiols without affecting disulfides. Importantly, cysteine sulfenic acids are especially unstable on denatured proteins and alkylated after denaturation to eliminate any cross reactivity.¹¹ After ascorbate reduction, any newly reduced thiols can react with either thiopropyl sepharose or biotin-HPDP for affinity enrichment and biochemical analysis. After resin enrichment, proteins are eluted by reduction of the disulfide-resin linkage and quantified by either Western blot or mass spectrometry. The biotin switch technique is thoroughly validated and widely used for indirect analysis of endogenous protein S-nitrosation.

Figure 1. Biotin Switch Technique (BST)

Organomercury Enrichment

More recently, several other chemoselective methods have emerged for S-nitrosothiol conjugation. Organomercury reagents react with thiols and nitrosothiols to form a stable mercury-thiolate conjugate(Figure 2).⁸ After complete alkylation of cellular thiols, organomercury resin or biotin-mercury conjugates allow direct enrichment of S-nitrosated proteins with no cross reactivity with disulfides. The mercury-linked proteins are then oxidized with performic acid to release modified proteins as observed with oxidized sulfonic acids. Since these probes are not commercially available, this method requires synthetic efforts using toxic mercury, warranting extreme safety precautions. Nonetheless, organomercury enrichment provides a highly sensitive and in-depth mass spectrometry profile of endogenous protein S-nitrosation from mouse tissues, allowing the identification of hundreds of endogenous target proteins and sites of S-nitrosation.¹⁴

Figure 2. Organomercury Enrichment

Phosphine Ligations

Organophosphine reagents have also been reported as robust probes for selective nitrosothiol conjugation (Figure 3).^5,9,10 Triarylphosphine probes lead to the chemo-selective reduction of S-nitrosothiols, initiated by the reaction of phosphine with the S-nitroso group to form an aza-ylide. Triarylphosphine probes have been designed to undergo a series of concerted reactions to yield different chemical linkages, including the bis-ligation with a triarylphosphine-thioester probe to yield a disulfide-iminophosphorane product.¹⁰ This probe is unique since it provides a direct linkage to both the originating nitric oxide nitrogen and the cysteine sulfur. While this approach was initially reported to detect S-nitrosated metabolites by mass spectrometry, it was later reported to achieve poor ionization efficiency in mass spectrometry-based proteomics analyses.^15,16 A separate triarylphosphine probe yields the one-step formation of a disulfide linkage at the site of S-nitrosation.⁹ Here the phosphine-thioester first forms a thiobenzamide adduct and thiolate followed by intermolecular thioester exchange with the released thiolates to generate a disulfide linkage. Altogether, SNO trapping by triarylphosphine (SNO-trap) methods enable robust enrichment and mass spectrometry profiling of native sites of S-nitrosation. Indeed, SNO-trap methods were used to identify differentially S-nitrosated proteins in a mouse model of Parkinson’s disease, revealing direct links to disease pathology.¹⁶

Figure 3. Phosphine Ligations

Sulfinic Acid Conjugation

S-Nitrosocysteine was first reported to react with phenylsulfinic acid to form a thiosulfonate linkage more than 30 years ago.¹⁷ Based on this reactivity, sulfinic acid probes have now been shown to modify sites of endogenous S-nitrosation on proteins, either by direct enrichment with biotinylated sulfinic acids or indirectly by exchange of the resulting thiosulfonate with a thiol-linked probe (Figure 4).^11,13 Sulfinic acids are unreactive towards cystine, oxidized glutathione, and the activated disulfide in Ellman’s reagent.¹¹ Furthermore, phenylsulfinic acid does not react with sulfenamides, which equilibrate to sulfenic acids in solution. Therefore, sulfinic acids exhibit privileged reactivity with S-nitrosothiols, yielding a stable thiosulfonate linkage. Similar to MMTS, thiosulfonates are electrophilic and react with free thiols to release methylsulfinic acid. In the thiosulfonate switch technique (TST), lysates are first alkylated with S-phenylsulfonylcysteine, which releases phenylsulfinic acid.¹³ Next, additional phenylsulfinic acid is added to the lysate, which converts nitrosothiols to thiosulfonates in acidic buffers. Finally, a thiol-linked biotin or fluorophore is added, which under acidic conditions reacts preferentially with thiosulfonates over any disulfides, yielding a more stable disulfide linkage at the former site of S-nitrosation.

Figure 4. Sulfinic Acid Conjugation

Conversely, S-nitrosated proteins can be directly conjugated to a probe in one step with biotin-conjugated sulfinic acids, such as the metabolite hypotaurine (biotin-SO₂H).¹¹ After complete alkylation with iodoacetamide, the lysate is incubated with biotin-SO₂H forming a thiosulfonate linkage at sites of endogenous S-nitrosation. Thiosulfonates are stable at neutral pH, providing a direct linkage for gel-based or mass spectrometry quantitation of endogenous S-nitrosation. Interestingly, MMTS alkylation prevents biotin-SO₂H conjugation, suggesting that methylsulfinic acid released by MMTS after thiol conjugation is present at sufficient concentrations to react at sites of S-nitrosation. While further studies are needed to explore this finding, it suggests that the ascorbate-dependent biotin switch technique may similarly lead to thiosulfonate formation after MMTS alkylation, which is then reduced by ascorbate. Finally, this reaction can be inverted to detect endogenous sulfinic acids on proteins, establishing a direct 1:1 stoichiometry in the reaction mechanism.¹¹ Overall, sulfinic acids and nitrosothiols provide a distinct biocompatible approach to profile the reciprocal reactivity of S-nitrosation and S-sulfination in biology.

Radical species are an unavoidable consequence of respiration and the environment and are tightly buffered by small molecule antioxidants and redox detoxifying enzymes. Aberrant oxidative signaling is perhaps one of the most important factors contributing to aging, neurodegeneration, heart disease, diabetes, and cancer. Now with several chemically orthogonal techniques, protein S-nitrosation can be readily analyzed by indirect detection of ascorbate sensitive thiols or by direct conjugation to organomercury, sulfinic acids, or triarylphosphine probes. The variety of S-nitrosation detection methods demonstrates the unique chemical properties of this reversible, electrophilic protein modification. With this emerging S-nitrosation toolset, multiple techniques can be simultaneously applied to corroborate endogenous S-nitrosation on select proteins by comparative proteomic profiling.

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