Antibody‐Free Detection of Protein Tyrosine Nitration in Tissue Sections

Tyrosine nitration is a covalent post-translational protein modification that occurs in vivo as a consequence of oxidative stress. It has been shown that tyrosine nitration is caused by peroxynitrite, which is formed by reaction between superoxide anions (O ) and nitric oxide (NO). Other reactive nitrogen species, such as NO2 + , NO2Cl, and NO2, can also be involved in tyrosine nitration. Recent studies have shown that 3-nitrotyrosine is a biomarker for several inflammatory diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis, and asthma. Tyrosine nitration has often been regarded as nonselective oxidative damage to proteins; however, recently more specific roles have been described. For example, tyrosine nitration triggers dissociation of IkB from nuclear factorkB (NF-kB); this results in the activation of a pathway that plays an important role in cancer and inflammation. The importance of tyrosine nitration in the pathology of multiple diseases and its potential role in signal transduction highlights the urgent need for reliable analytical methods to study protein nitration. Several methods have been developed for the detection of nitrotyrosine; these employ antibodies, mass spectrometry, or fluorogenic tagging. Currently, tyrosine-nitrated proteins in tissue sections are detected by immunohistochemistry with antibodies directed at nitrotyrosine. Protein-bound nitrotyrosine in tissue homogenate and blood plasma is usually detected by immunoblot methods; these also rely on nitrotyrosine-directed antibodies. However, nitrotyrosine antibodies have disadvantages, such as a lack of selectivity for nitrotyrosine, high background signals in certain tissues, high costs, and limited stability upon prolonged storage. In this study, we introduce a novel method that does not rely on antibodies to detect nitrotyrosine in biological samples. This method involves two reaction steps to convert the 2-nitrophenol functionality of protein-bound nitrotyrosine chemoselectively into a fluorophore (Scheme 1). In the first reaction step, the 2-nitrophenol functionality is reduced to a 2-aminophenol functionality by sodium dithionite. In the second step, the 2-aminophenol functionality is converted into a fluorophore by reaction with salicylaldehyde and Al . This novel histochemical staining selectively detects 2-nitrophenol, as well as 2-nitrosophenol and 2-aminophenol functionalities. In this study, we describe the application of this methodology to detect nitrotyrosine in tissue sections by fluorescence microscopy. This method is also applicable for staining protein-bound 2-nitrophenol functionalities on Western blot membranes in complex biological samples such as blood plasma. Initially, we studied the fluorescence of the Schiff base of 2aminophenol and salicylaldehyde complexed with aluminium. The fluorescence spectra of this complex in solution showed an excitation wavelength (lex) of 412 nm and an emission wavelength (lem) of 520 nm in H2O and in MeOH (Figure S1 and S2 in the Supporting Information). The fluorescence proved to be linear between 0.31 and 10 mm in both solvents (Figure S3), and the fluorescence signal in H2O was eight times stronger than that in MeOH. Thus, H2O is the preferred solvent for the detection of nitrotyrosine in biological samples. The first reaction step for the histochemcial staining (Scheme 1) is reduction of 2-nitrophenol by a freshly prepared solution of sodium dithionite (Na2S2O4) in demineralised water. This reagent provides fast and complete conversion of 2-nitrophenol in an aqueous environment, thus demonstrating that sodium dithionite is an appropriate reducing agent for this reaction. Several control experiments were performed for the second staining reaction (Scheme 1), the formation of the Schiff base between the 2-aminophenol functionality and salicylaldehyde in complex with Al . Analysis of the fluorescence spectra of the reagents in the solution used for this step (containing salicylaldehyde and aluminium) gave very weak fluoresScheme 1. Reduction of a protein bound 2-nitrophenol functionality to a 2aminophenol functionality, and subsequent conversion into a fluorophore. a) aqueous Na2S2O4, b) aqueous AlCl3 and salicylaldehyde.