Sinapinic acid can replace ascorbate in the biotin switch assay

Abstract

Background

Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.

Methods

The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO₂^- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.

Results

We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.

Conclusions

Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.

General significance

The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay.

Introduction

Protein S-nitrosation is an important post-translation modification that affects various proteins involved in number of cellular processes, mediated by nitric oxide (NO) through redox-dependent conversion of cysteine thiols of proteins to S-nitrosothiols [1], [2], [3], [4]. Earlier studies have shown that modifying cysteine residues of proteins may result in altered protein function thus nitrosothiols play major roles in human health and disease [5].

A major contribution to the field of S-nitrosothiols is the biotin switch assay, introduced by Jaffrey et al. in 2001 [6], [7]. In this assay, proteins are denatured by sodium dodecyl sulphate (SDS) in the presence of methyl methane thio-sulfonate (MMTS) to block free thiols. Thiols present in the interior of the protein are exposed when they are incubated with SDS at 50 °C. After acetone precipitation to remove excess of MMTS, 1 mM ascorbate and biotin-HPDP are added to maintain the Cu (I)-catalyzed reduction of the nitrosothiols and to label the reduced thiol with biotin. Proteins are separated by non-reducing SDS-PAGE followed by immunoblotting. Biotinylated proteins are detected using streptavidin-HRP or anti-biotin HRP and chemiluminescence detection system. This assay is first method to identify S-nitrosated proteins on a gel, which enables their subsequent isolation and identification by techniques like mass spectrophotometry.

Many researchers have modified the biotin switch assay either by altering the concentration of ascorbate or the incubation times for the various steps, or adding metals or metal chelators. Several reports indicate that the high levels of ascorbic acid utilized for denitrosation can also reduce disulfide bonds in proteins thus leading to “false” biotinylation of proteins that have no S-nitrosothiols [8], [9]. A recent article by Gladwin and coworkers indicated that the extent of biotin-labelling depends upon factors such as buffer composition and choice of the metal-ion chelators. Other modifications of the biotin switch method include the addition of trace amounts copper to increase the sensitivity of the assay without compromising its specificity [10].

Sinapinic acid (3, 5-dimethoxy-4-hydroxy cinnamic acid) is widely used as matrices in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [11]. Previous studies with sinapinic acid have shown that it reacts with peroxynitrous acid at neutral pH to yield a red product, O-nitrososinapinic acid. The O-nitrososinapinic acid is a direct analog of S-nitrosothiols and was shown to be capable of transnitrosating thiols [12]. However, O-nitroso intermediates were unstable, which would lead to their dimerization via the formation of mono-lactone like products at pH 7.4 [13], [14].

The objectives of our study were to determine if sinapinic acid could: (1) denitrosate S-nitrosothiols; and (2) to test if sinapinic acid could replace ascorbic acid as the denitrosating agent in biotin switch assay.