Detection of Protein S-Nitrosylation with the Biotin Switch Technique (original) (raw)

. Author manuscript; available in PMC: 2011 Jun 22.

Abstract

Protein _S_-nitrosylation, the post-translational modification of cysteine thiols to form _S_-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for _S_-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during physiological and pathophysiological conditions. The focus of the current review is the biotin switch technique (BST), which has become a mainstay assay for detecting _S_-nitrosylated proteins in complex biological systems. Potential pitfalls and modern adaptations of the BST are discussed, as are future directions for this assay in the burgeoning field of protein _S_-nitrosylation.

Keywords: Biotin switch technique, _S_-nitrosylation, _S_-nitrosothiol, redox, cysteine, thiol, nitric oxide, nitrosative stress

BASICS OF S-NITROSYLATION

Protein _S_-nitrosylation—the covalent adduction of a nitroso group to a cysteine thiol side chain—has recently emerged as a principle mechanism by which nitric oxide (NO) mediates a wide range of cellular functions and phenotypes [1, 2]. _S_-nitrosylation regulates diverse pathways such as G-protein-coupled receptor signaling [3–5], death receptor-mediated apoptosis [6–11], glutamate-dependent neurotransmission [12–15], vesicular trafficking [16–19], stimulation of prostaglandin synthesis [20–22], and the unfolded protein response [23]. In addition, aberrant _S_-nitrosylation is implicated in disease states such as tumor initiation and growth [24–28], neurodegeneration [23, 29–32] and malignant hyperthermia [33]. Consequently, much effort is focused on understanding the role of _S_-nitrosylation in normal physiology and its contribution to pathophysiology. For example, several recent studies have shown that dysregulated _S_-nitrosylation of the ryanodine receptor (Ca2+-release channel) may contribute to cardiac arrhythmias [34], heat stroke [33] and impaired exercise capacity [35]. As scientific interest in protein _S_-nitrosylation continues to intensify, an increasing number of studies are relying on the biotin switch technique (BST) for the detection of endogenously _S_-nitrosylated proteins (protein-SNOs). The introduction of this assay by Jaffrey et al. in 2001 [36] has served as an impetus for studies probing _S_-nitrosylation in vivo, largely due to its superb compatibility with ubiquitous molecular methods (e.g. SDS-PAGE, immunodetection, mass spectrometry).

NO- VS. SULFUR-BASED ASSAYS OF S-NITROSYLATION

The sulfur-nitrogen bond of an SNO is particularly labile and can undergo both homolytic and heterolytic cleavage reactions [37, 38]. The lability of the S-NO bond has served as the cornerstone for numerous SNO detection strategies, though the chemistries employed following SNO cleavage differ greatly between assays (Fig. 1). Most techniques detect the NO or nitrite (NO2−) liberated upon S-NO cleavage, and hence can be considered “NO-based” strategies. In these assays, divalent mercury (e.g. HgCl2) is often employed to heterolytically cleave the S-NO bond, producing a mercury-thiol complex and nitrosonium ion (NO+); the latter is a potent nitrosant and undergoes rapid hydration to NO2− at neutral pH. Techniques (spectrophotometric or fluorescent) that detect the NO2− product include the Saville [39–41], diaminonapthalene [39, 42] and diaminofluorescein assays [42–45].

Fig. 1.

Fig. 1

A general comparison of NO- and sulfur-based strategies for detecting protein _S_-nitrosylation. As an example, three lysates containing various amounts of protein _S_-nitrosylation are subjected to both NO- and sulfur-based assays. NO-based strategies include the Saville and diaminofluorescein (DAF) assays, which employ a chemical probe, and Hg-coupled photolysis-chemiluminescence (PCL), which detects NO gas liberated by SNO homolysis and can differentiate SNO from metal-NO. Importantly, this assay is highly sensitive (low nanomolar SNO concentrations can be detected) and has been well-validated with genetic models of disrupted NO/SNO metabolism [108, 109]. It therefore serves as a standard method for probing _S_-nitrosylation in vivo. With a complex biological sample (e.g. a lysate), these NO-based strategies can readily determine the absolute amount of SNO per sample, but cannot readily detect an individual protein-SNO. A sulfur-based strategy, such as the biotin switch technique (BST), employs covalent “tagging” at the sulfur atom of each SNO, thus facilitating relative quantitation and protein-SNO identification.

Another common NO-based technique employs homolytic or reductive conditions to cleave the S-NO bond, followed by chemiluminescent detection of the liberated NO via reaction with ozone. Such methods include Hg-coupled photolysis-chemiluminescence [46, 47] and the copper-cysteine-carbon monoxide (3C) assay [48–50]. Though each of these NO-based methods is well suited for SNO quantitation (relative to SNO standards), they have limited use in functional studies of _S_-nitrosylated proteins within complex mixtures because the proteins of interest must be purified (e.g. by immunoprecipitation) prior to SNO measurement. While this method has been applied successfully in a number of cases—including _S_-nitrosylated hemoglobin [51–53], caspase-3 [11, 54], thioredoxin-1 [55], c-Jun N-terminal kinase [56], G-protein-coupled receptor kinase 2 [5], ryanodine receptor [57, 58] and prokaryotic OxyR [59]—the arduous nature of the approach has limited its application.

In contrast to NO-based assays, the BST is unique in that it targets the sulfur atom of an SNO without regard for the fate of any liberated NO species; it can thus be considered a “sulfur-based” strategy. As the BST employs covalent “tagging” of protein-SNOs, it can detect individual protein-SNOs in a complex mixture (since the tag is added to the protein of interest). For studies of a specific protein or class of proteins, the BST has therefore proved to be of great utility (> 200 publications to date).

As described above, the major differences between NO- and sulfur-based strategies for SNO detection are summarized in Fig. 1. Importantly, the type of information gained from either approach is different. NO-based assays are capable of absolute quantitation (e.g. 10 nmol SNO per 1 mg protein), though they do not generally discriminate the source of each protein-SNO. In contrast, the BST tends to be more qualitative (relatively quantitative); its great advantage is its ability to detect individual protein-SNOs in a complex mixture, as well as to identify novel protein-SNOs.

WORKFLOW OF THE BIOTIN SWITCH TECHNIQUE

As illustrated by Fig. 2, the BST consists of three principal steps: 1) “blocking” of free cysteine thiols by _S_-methylthiolation with MMTS (a reactive thiosulfonate); 2) conversion of SNOs to thiols via transnitrosation with ascorbate; and 3) in situ “labeling” by _S_-biotinylation of the nascent thiols with biotin-HPDP, a reactive mixed disulfide of biotin. The degree of biotinylation (and thus _S_-nitrosylation) is determined by either anti-biotin immunoblotting or streptavidin pull-down followed by immunoblotting for the protein(s) of interest.

Fig. 2.

Fig. 2

Overview of the biotin switch technique. In the example shown, three lysates with various degrees of _S_-nitrosylation are subjected to the assay. The “blocking” step involves _S_-methylthiolation of each cysteine thiol with _S_-methylmethanethiosulfonate (MMTS). Next, ascorbate is employed to convert each SNO to a free thiol via a transnitrosation reaction to generate _O_-nitrosoascorbate. In the “labeling” step, each nascent free thiol (previously an SNO site) is biotinylated with biotin-HPDP. Biotinylated proteins are enriched by avidin affinity media, and analyzed by SDS-PAGE/immunoblotting. Total protein-SNOs or an individual protein-SNO can be detected with avidin-HRP or with an antibody against a protein of interest, respectively. As illustrated, the degree of pulldown correlates with protein _S_-nitrosylation. Prior to avidin pulldown, a small fraction of each sample is analyzed to determine protein “input.”

The blocking step is typically initiated by the addition of SDS and MMTS, followed by heating at 50 °C and separation of proteins from excess MMTS by acetone precipitation. The combination of heat and SDS functions to fully denature proteins, thus granting MMTS optimal access to natively buried protein thiols. Effective blocking of free thiols is required to minimize “background” biotinylation (i.e. biotinylation that results from incomplete blocking) and to maximize assay sensitivity.

The second step in the BST converts the SNO to a free thiol so that it can be biotinylated. This is achieved by allowing ascorbate—a di-enol antioxidant with unique reactivity towards SNOs—to undergo a transnitrosation reaction with the protein-SNO. As initially reported by Holmes and Williams [60], this reaction proceeds via a concerted nitroso transfer mechanism whereby the SNO and ascorbate are converted to thiol and _O_-nitrosoascorbate. Notably, ascorbate is several orders of magnitude more reactive than other _N_- or _O_-based nucleophiles (e.g. amines, phenols) [61, 62]. The reaction between SNOs and ascorbate also exhibits a critical pH-dependence and is favored by transition metal chelation: alkaline conditions promote the nitroso transfer reaction, likely by increasing the fraction of mono-deprotonated (i.e. nucleophilic) ascorbate, whereas transition metals may enable alternative chemistry (see below). The specificity of the BST for protein-SNOs is predicated on the fact that ascorbate will convert SNOs to free thiols without reducing other cysteine-based oxidations such as _S_-glutathionylation or _S_-oxides (sulfenic, sulfinic and sulfonic acids). This specificity is supported by thermodynamic measurements (discussed below) and by several experimental validations [36, 63].

The third step of the BST involves biotinylation of the nascent thiol (i.e. previous SNO site). This is performed concomitantly with the ascorbate reaction, such that the newly liberated free thiols are immediately biotinylated. Excess biotin-HPDP is removed by acetone precipitation, and protein biotinylation (i.e. previous _S_-nitrosylation) is assessed by one of multiple routes. Avidin-agarose is frequently employed to enrich the biotinylated proteins, followed by elution and SDS-PAGE. It is important to note that the biotin tag is attached via a disulfide linkage, and therefore reductants (e.g. DTT) will remove the tag. This reductive elution strategy is frequently employed to elute biotinylated proteins from avidin-agarose prior to SDS-PAGE, especially when the biotin-tag is not employed for immunodetection (e.g. when blotting an individual protein). As demonstrated in Fig. 3A, the BST readily detects _S_-nitrosylated GAPDH in cytokine-stimulated macrophages, in which inducible nitric oxide synthase (iNOS) is upregulated [64–66]. Alternatively, total protein-SNOs can also be assessed by non-reducing SDS-PAGE and immuno-detection of the biotin tag. As shown in Fig. 3B, this approach easily detects total protein-SNOs in HEK293 cells following treatment with _S_-nitrosocysteine (CysNO).

Fig. 3.

Fig. 3

A typical BST detects both endogenous and exogenous _S_-nitrosylation in cultured mammalian cells. A) Murine RAW264.7 macrophages were either untreated or cytokine-stimulated with lipopolysaccharide (500 ng/ml) and IFN-γ (100 U/ml) for 16 h, which drives NO production. Cellular extracts were subjected to the BST and probed for _S_-nitrosylated GAPDH (GAPDH-SNO), along with ascorbate and pre-photolysis controls. Notably, omission of ascorbate leads to nearly complete loss of biotinylation and pre-photolysis with a Hg vapor lamp greatly attenuates the same signal. B) Whole cellular protein-SNOs are detected by treating HEK293 cells with 200 µM _S_-nitrosocysteine (CysNO) for 10 min. Cellular extracts were subjected to the BST and 5% of each biotinylation reaction (~ 40 µg) was analyzed by immunoblotting with avidin-HRP and anti-GAPDH antibody (for “input”).

It is important to note that the BST can be used to assay _S_-nitrosylation by constitutive NOS isoforms (nNOS and eNOS; Fig. 4A) in addition to iNOS, and to examine _S_-nitrosylation dynamics in the context of signal transduction, as demonstrated for G-protein-coupled receptor kinase 2 (GRK2) following stimulation of multiple G-protein-coupled receptors (Fig. 4B). Thus, when performed appropriately it is highly sensitive as well as specific. Further, the BST can be used to identify the NOS isoform(s) involved (Fig. 4A and C), and to reveal the participation of denitrosylase activities or endogenous _S_-nitrosylating agents (Fig. 4D) across a wide range of cellular stimuli. Collectively, these examples illustrate the utility of the BST for assessing _S_-nitrosylation in vivo, particularly when used alongside the methodological, pharmacological and genetic tools demonstrated in Figs. 3 and 4.

Fig. 4.

Fig. 4

The BST can be combined with pharmacological and genetic tools to study _S_-nitrosylation in primary cells and tissues in vivo. A) GRK2 is _S_-nitrosylated in HEK293 cells that stably overexpress the constitutive NOS isoforms (eNOS and nNOS). B) G protein coupled receptor (GPCR)-specific agonists (ATP, adenosine triphosphate; BK, bradykinin; ISO, isoproteronol; CTL, control), each of which leads to eNOS activation, increase GRK2 _S_-nitrosylation in human umbilical vein endothelial cells. C) GRK2 _S_-nitrosylation is diminished in lungs from eNOS−/− mice relative to wild type (WT) mice. D) GRK2 _S_-nitrosylation is increased in lungs from mice lacking a major SNO-metabolizing enzyme, GSNO reductase (GSNOR−/−). Figure adapted from reference [5].

PRE-PHOTOLYSIS AS AN INDEPENDENT STRATEGY IN THE BST

There is little doubt that the signals arising from a BST following treatment with CysNO (Fig. 3B) represent bona fide _S_-nitrosylated proteins. Like all other biological assays, however, internal and external controls should be employed to verify data obtained with the BST, particularly when assaying endogenously _S_-nitrosylated proteins. These approaches frequently include: 1) omission of ascorbate during the labeling step of the BST; 2) overexpression or knockdown of a particular NOS isoform; and/or 3) pharmacological activation or inhibition of NOS. Though each of these approaches has been consistently applied to assay SNOs by the BST, we recently introduced an independent technique that involves photolysis of the SNO prior to performing the BST (dubbed “pre-photolysis”) [5, 63]. This methodology—a UV-based homolysis of the S-NO bond—is a simple and specific tool for assaying SNOs with the BST, particularly under conditions where NOS activation or inhibition are either unfeasible (e.g. human patient samples) or ineffective (e.g. NOS activity-independent, a characteristic of signaling activated by denitrosylation). This approach is based on Hg-coupled photolysis-chemiluminescence, in which the Hg-displaceable pool of NO groups (i.e. SNO) is photolyzed and detected via reaction with ozone. Photolysis eliminates SNO. Consequently, pre-photolysis serves as a complementary approach to BST. As shown in Fig. 3A, the signal from endogenously _S_-nitrosylated GAPDH is attenuated by a brief exposure to high intensity UV light. A schematic illustration in Fig. 4 shows how pre-photolysis and ascorbate can be combined to rigorously assess a complex sample for protein _S_-nitrosylation.

DESIGN OF THE PRE-PHOTOLYSIS APPARATUS

Pre-photolysis is performed by exposing the sample(s) to a strong UV radiation source within a light-sealed reaction chamber. Overall such an apparatus should: 1) deliver high intensity radiation at 335 nm (the ideal wavelength for S-NO photolysis [47, 67, 68]); 2) prevent heating of the sample to avoid thermolytic reactions; 3) allow multiple samples to be photolyzed simultaneously; and 4) protect the operator from any harmful UV exposure. We employ a 200 watt, 1.9 amp Hg vapor lamp (Ace Glass, catalog # 7825-32), which delivers approximately 50% of its energy in the ultraviolet region. This lamp, along with an appropriate power supply, can be fitted into a sealed photochemical reaction cabinet (e.g. Ace Glass, catalog # 7836-20). Samples are generally photolyzed in borosilicate glass vessels (Pierce catalog # 13504). Typically a two-minute exposure approximately 3 cm from the lamp is sufficient to photolyze at least 50% of a protein-SNO.

Several more economical sources of UV radiation may also suffice for pre-photolysis, though they are likely less efficacious than a Hg vapor lamp. These include a UV transilluminator (often used to visualize DNA with ethidium bromide staining) and a UV crosslinker that provides radiation with wavelengths greater than 300 nm. In the latter case, a suitable apparatus would be the Ultraviolet Products (UVP) CL-1000 crosslinker (UVP, catalog # 81-0112-01) fitted with five long-range UV lamps (UVP, catalog # 34-0006-01).

METHODOLOGICAL ISSUES AND CONCERNS RAISED ABOUT THE BST

Despite its widespread use, the BST is technically challenging and labor intensive. Since each step contains potential sources error, a rigorous experimentalist will include proper negative and positive controls to add confidence to their results. In the case of _S_-nitrosylation, numerous tools can be employed to check assay efficiency and to manipulate the stability or formation of protein-SNOs, thus adding great confidence to results obtained with the BST.

When comparing protein-SNOs across multiple conditions, it is imperative that each sample contains equivalent protein abundance, or “inputs”. While some degree of normalization can be allowed, linearity (with respect to protein input and biotinylation) of the BST has not been demonstrated. Since proteins are subjected to multiple acetone precipitation steps and pellet washes, SDS-PAGE analysis should be performed on the biotinylated material prior to avidin pulldown. The final step of the BST is, for all intents and purposes, an affinity pulldown, which are almost always reported in the literature as both an “input” and a “pulldown.” The BST should be held to the same standards.

The blocking step of the BST can also present a technical challenge, as some protein thiols can be resistant to complete blocking, resulting in high levels of SNO-independent biotinylation. In the absence of proper controls, incomplete blocking can be problematic since it may lead to misinterpretation of the data (i.e. “false-positive” signal). However, if performed correctly (e.g. use of ascorbate), ineffective blocking will lower assay sensitivity (increase signal to noise) rather than specificity. Conversly, reverse strategies such as omitting ascorbate or employing pre-photolysis can be used to determine the degree of blocking. Both of these treatments will attenuate a true SNO signal. Further, the blocking reaction can be lengthened to improve efficiency, though SNO stability may be compromised at 50 °C due to thermolysis.

The specificity of the BST for SNOs (versus other cysteine-based modifications) had been questioned in several works. Here we review the actual data in these publications, and explain why observations are more likely artifact than source of real concern. Landino et al. suggested that ascorbate reduces tubulin disulfides, a claim the authors use to challenge the validity of the BST [69]. However, as discussed below, reduction of alkyl (biological) disulfides by ascorbate is highly unfavorable thermodynamically, and an explanation for how “disulfides” were purportedly reduced was not provided. These authors in fact employed high concentrations of peroxynitrite to “oxidize” tubulin, though peroxynitrite also nitrosates thiols (albeit at low yields) thereby generating SNOs [70–72]; it is likely that _S_-nitrosylated tubulin was generated under these conditions. A later report by Huang et al. argued that the BST yields “artifactual” ascorbate-dependent biotinylation of native reduced BSA [73] (i.e. SNO-independent). The authors did not provide a mechanistic basis for their observation, and subsequent work has reproduced the findings of Huang, which was caused by a window light artifact (BST should be performed in the dark) [63].

Most recently Guisitarini et al. have challenged the BST mainly on the grounds that ascorbate reduces DTNB [74]. However, this well known reaction has no bearing on reduction of biological (alkyl) disulfides: DTNB is a highly electrophilic, aryl disulfide that readily undergoes both hydrolysis and homolysis, the latter facilitated by ascorbate [75, 76]. DTNB is therefore not representative of biological disulfides or in vivo Cys-based oxidation products. More perplexing, however, is these authors’ suggestion that ascorbate reduces low molecular weight biological disulfides (e.g. glutathione, homocysteine and cysteine disulfide) and protein mixed disulfides, but not intramolecular protein disulfides (under denaturing conditions). In fact, none of these reactions are favored thermodynmically, and there is no kinetic or thermodynamic basis for the difference between mixed and intramolecular protein disufides under denaturing conditions. On closer inspection, yield of reaction with cystine and glutathione disulfide was extremely low and appeared to saturate at a few percent over several hours, consistent with the presence of a contaminant (e.g. contaminant metals) or other artifact. Further, the authors measured disulfide reduction in the continuous presence of fluorescent thiol alkylators (bromobimanes), which irreversibly pull reactions by Le Chatelier’s principle, and treated other samples with DTT to prevent “possible re-oxidation of thiols after their reduction by ascorbate” [74]. These approaches, which would artifactually generate thiols, cast doubt on the conclusions. These authors also do not provide a mechanistic explanation for their results or reference prior work to the contrary [77–81]. But most important of all, they do not perform the BST, whereas a comparative analysis of the BST across cell lysates derivatized by _S_-glutathionylation, _S_-oxidation and _S_-nitrosylation revealed that only the _S_-nitroylated proteins were detected [63].

Shared between all these claims of “artifactual signals” is the notion that ascorbate can reduce alkyl disulfides. However, this concept is both unsupported and difficult to reconcile experimentally with the use of MMTS as a blocking agent, which converts protein thiols to mixed methyl disulfides. Clearly the BST would never work if ascorbate removed these “blocked” methyl disulfides. Further, the two electron standard reduction potential of cysteine disulfides is −170 to −320 mV [77], indicating that this endergonic reaction will not likely couple to the two electron oxidation of ascorbate, which is also unfavorable (the standard reduction potential of dehydroascorbate to ascorbate is +70 mV [82]). Thus, one would not expect ascorbate to directly reduce any biological protein cysteine oxidation products (except highly transient and unstable thiyl or sulfinyl radicals). In contrast, these electrochemical measurements favor the reverse reaction—thiol-dependent reduction of dehydroascorbate to ascorbate—a scenario supported by extensive in vitro and in vivo experimentation [78, 80, 81]. The same thermodynamic arguments hold for higher _S_-oxides as well [77].

Although the BST is highly specific for protein SNOs [63], we have noted that the presence of indirect sunlight (from an adjacent window) during the labeling step closely recapitulates the artifactual results of Huang et al. [73]. This SNO-independent biotinylation reaction is fully reversed by DTT, confirming that indirect sunlight indeed promotes an artifactual ascorbate-dependent protein-biotin disulfide during the labeling step. Under these conditions, biotin-HPDP (a synthetic aryl disulfide) undergoes quantitative reduction to biotin-thiol, though ascorbate exhibited no measureable reactivity in the absence of sunlight [63]. This production of high micromolar biotin-thiol during the BST would lead to artifactual protein biotinylation via thiol/disulfide exchange with “blocked” (i.e. _S_-methylthiolated) proteins. The mechanism of this side reaction may involve homolysis of biotin-HPDP to generate an unstable alkyl thiyl radical of the biotin group and a resonance-delocalized aryl radical of 2-thiopyridine. In the presence of ascorbate, these thiyl radicals would be reduced to thiol [77] and therefore contribute to the observed artifact. Importantly, similar reactions between light, aryl disulfides and ascorbate have been reported [75, 83]. These issues underscore the critical need to perform the labeling step of the BST in a light-free environment, and to employ metal chelators (more below).

POTENTIAL PROBLEMS ASSOCIATED WITH EXOGENOUS METALS IN THE BST

Ascorbate will drive one-electron reductions of Cu2+ and Fe3+ ions, which may result in reactions that compromise BST specificity, including production of ascorbate and hydroxyl radicals. These reactions are readily mitigated through the use of metal chelators (e.g. EDTA, DTPA, neocuproine) [84–87]. Despite the widespread application of metal chelation with the BST, one recent report suggests a metal-dependence for the BST to efficiently detect SNOs [88]. However, this conclusion overlooks the different chemistry operative in the presence and absence of transition metals: transition metals favor reductive chemistry by ascorbate over transnitrosation. Thus at low concentrations of ascorbate (<1 mM) as employed by Wang et al. [88], transition metals promote direct SNO reduction (metals serve as catalysts). Conversely, transnitrosation reactions, which form the basis of BST specificity are favored by metal sequestration and higher ascorbate concentrations [60]. Increasing the amount of ascorbate (5 – 50 mM ascorbate) should remedy the reported “requirement” for exogenous metals that may increase assay sensitivity, but at the expense of specificity. In particular, redox active metals are known to promote manifold reactions (both ascorbate dependent and independent), such as Cu2+-mediated thiol oxidation [89–91] and Fenton chemistry [92–94]. Given the potential for such side reactions to compromise assay specificity, the addition of exogenous redox active metals in the BST cannot be advocated without more rigorous validation in complex biological systems.

Given the known reactivity of Hg towards thiols and SNOs, numerous studies have attempted to employ Hg salts (e.g. HgCl2) to quench SNOs prior to performing the BST (and thus serve as an internal assay control in lieu of omitting ascorbate). Despite the frequent application of Hg salts in NO-based assays (described above), the high affinity of Hg salts for free thiols [95–97] makes this strategy inherently problematic for the BST. As nicely demonstrated by Zhang et al. [98], HgCl2 prevents biotinylation of free thiols and lowers any signal irrespective of _S_-nitrosylation status. Since all sources of biotinylation (i.e. signals) in the BST are attenuated by Hg salts, it is unclear how such a strategy could be used to distinguish an SNO from a false positive signal due to incomplete blocking; therefore, Hg salts should not be used in lieu of other well-validated controls (e.g. pre-photolysis, NOS inhibition, omitting ascorbate). By contrast, in an NO-based assay such as photolysis-chemiluminesence, Hg-thiol reactions identify the source of NO with Cys thiol by quenching the signal.

MODERN PERMUTATIONS AND ADAPTATIONS OF THE BST

Various groups have adapted the BST to suit specific experimental goals. For example, several labs have trypsinized the biotinylated material prior to avidin pulldown to allow mass spectrometry-based determination of protein-SNO sites (i.e. specific cysteine residues targeted by _S_-nitrosylation) [5, 99–101]. In addition, a thiol-reactive hexahistidine tag has been used in lieu of biotin-HPDP to further facilitate mass spectrometric analysis [102]. These studies have further demonstrated the utility of the BST for SNO-oriented proteomics and bioinformatics. However, some of these strategies rely on a single peptide (containing the SNO site) for the identification of an entire protein, and thus stringent mass spectrometric criteria must be applied to these approaches to avoid incorrect protein (and SNO site) identification.

In another novel approach, Kettenhofen et al. substituted biotin-HPDP with thiol-reactive cyanine dyes to combine in-gel fluorescent detection strategies with the BST [103, 104]. Han et al. similarly employed coumarin-based fluorophores to the same end [105]. A major advantage of this approach is that it is readily adaptable to 2-dimensional differential in-gel electrophoresis (2D-DIGE) techniques where multiple differentially fluorescent-labeled samples are co-analyzed on a single 2D electrophoresis gel [106, 107].

TYPICAL PROTOCOL FOR THE BIOTIN SWITCH TECHNIQUE

Fig. 5.

Fig. 5

An illustration employing UV pre-photolysis and ascorbate controls within the BST for the analysis of a single lysate. One sample is split to two fractions, which are either untreated or exposed to a strong UV light source (“pre-photolysis”) prior to performing the BST. The pre-photolysis step leads to homolytic cleavage of the S-NO into NO and an unstable thiyl radical, which is either reduced to a thiol or oxidized to a higher _S_-oxide. In either case, the signal from an SNO will be attenuated by pre-photolysis, while free thiols or disulfides are unaffected. Inclusion or exclusion of ascorbate during the labeling step of the BST can also be employed to assay for protein-SNOs. This approach, which employs internal controls (e.g. pre-photolysis and ascorbate), is ideal when external controls such as NOS inhibition or activation are not feasible (e.g. human tissue samples) or ineffective (as in the case of reactions regulated by denitrosylation).

ACKNOWLEDGEMENTS

The authors would like to thank Alfred Hausladen and Abhijit Chakladar for their thoughtful comments and technical expertise. This work was supported by the National Institutes of Health grant P01-HL075443.

Footnotes

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References