Original Contribution
Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

https://doi.org/10.1016/j.freeradbiomed.2004.12.012Get rights and content

Abstract

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.

Introduction

S-Nitrosation of cysteine residues in target proteins has been suggested as a posttranslational modification that plays an important role in nitric oxide (NO)-mediated, cyclic guanosine monophosphate-independent, cellular events. A broad spectrum of proteins, for example, hemoglobin, N-methyl-D-aspartate receptor, p21ras, caspase-3, and I-κB kinase [1], [2], [3], [4], [5], [6], have been found to undergo S-nitrosation at their active cysteine residues, resulting in changes of their activities. Most previous studies were conducted on purified proteins, or focused on the modification of an individual protein in NO or S-nitrosothiol-treated cells or tissue extracts. In order to fully understand how S-nitrosation is involved in NO-mediated biological processes, it is essential to examine protein S-nitrosation at the whole-cell level and to identify the proteome of S-nitrosated proteins.

Recently the biotin-switch assay, developed by Jaffrey et al. [7] has been used to identify S-nitrosated proteins in tissues, cells and mitochondria [7], [8], [9], [10]. This assay involves three sequential steps: blockage of the cellular free thiols by methyl methanethiosulfonate (MMTS), specific reduction of S-nitrosothiols to thiols by ascorbate, and labeling the nascent thiol with biotin. For Western blot analysis, biotin-labeled proteins can be easily detected by an anti-biotin antibody [7] or streptavidin-peroxidase [9]. Combined with proteomic approaches, the assay can be used to identify cellular S-nitrosated proteins.

Although the biotin-switch assay has been used in a variety of experiments, the mechanistic basis of this assay has not yet been fully established. As noted above, this assay is dependent upon the ability of ascorbate to reduce S-nitrosothiols. An implicit assumption of this assay is that all S-nitrosated proteins have similar reactivity toward ascorbate, so that the detected species represent the proteins that are sensitive to S-nitrosation, rather than the S-nitrosated proteins that are sensitive to ascorbate reduction. In addition, the efficiency of ascorbate reduction will determine the overall sensitivity of this assay. However, it has been shown that the reaction of ascorbate with low-molecular-weight S-nitrosothiols (for example, S-nitrosoglutathione, GSNO) is slow (k = 0.05 M−1 s−1) , and different S-nitrosothiols tend to have different reactivities [11], [12], [13]. In this study we have examined the effect of S-nitrosothiol and ascorbate concentrations, as well as reaction time on Western blot analysis. The sensitivity of the biotin-switch assay was compared to the tri-iodide-based chemiluminescence method of S-nitrosothiols detection. The biotin-switch assay was applied in proteomic identification of cellular S-nitrosothiols when RAW264.7 cells were treated with S-nitrosocysteine (CysNO).

Section snippets

Materials

l-Cysteine, sodium nitrite, diethylenetriamine pentaacetic acid (DTPA), N-ethylmaleimide (NEM), streptavidin agarose, sodium ascorbate, 2-mercaptoethanol (2-ME), neocuproine, and sodium dodecyl sulfate (SDS) were purchased from Sigma. Hepes, sodium chloride and Triton X-100 were obtained from Fisher. N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (biotin-HPDP) and enhanced chemiluminescence (ECL) reagents were from Pierce. Anti-biotin (goat) peroxidase conjugate was from Calbiochem.

Decomposition of S-nitrosothiols in the presence of ascorbate

The key step in the biotin-switch assay is the reduction of S-nitrosothiols by ascorbate to form thiols. Therefore the efficiency of this reaction should determine the sensitivity and specificity of this assay. It has been shown that ascorbate can reduce GSNO to GSH and nitrite/nitrate and the rate of decay is slow at physiological pH [11], [12], [13]. Our examination of the ascorbate-mediated S-nitrosothiol decay rate gives rate constants of 0.05 M−1 s−1 for GSNO and 0.0068 M−1 s−1 for SNAP at

Discussion

Recently the biotin-switch assay has been successfully applied in proteomic studies to identify cellular S-nitrosated protein [7], [8], [9], [10]. This assay involves three major steps: blockage of free thiols, specific reduction of S-nitrosothiols to thiols by ascorbate, and labeling the nascent thiols with biotin groups. MMTS [7] and NEM [19] have been used to block thiols, giving a clean background in nontreated samples. Biotin-HPDP can efficiently label free thiol groups. Therefore the

Acknowledgments

This work is supported by grant GM55792 from the National Institute of General Medicine and predoctoral fellowship 0310032Z from the American Heart Association. We would also like to thank Shaun Summerill for technical assistance.

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