Filter blot method: A simple method for measuring 3-nitrotyrosine in proteins of atmospheric particulate matter

Air pollutants, such as nitrogen dioxide (NO₂), ozone (O₃), and particulate matter (PM), have been epidemiologically reported to contribute to the onset and exacerbation of asthma. We have previously shown that several proteins in atmospheric PM are allergenic in mouse asthma models and that these proteins are nitrated by atmospheric NO₂ and O₃ in chemical reactions. Based on these results, the amount of 3-nitrotyrosine (3-NT) in atmospheric PM could be an air pollution marker integrating NO₂, O₃, and PM. We established a method to measure 3-NT by high-performance liquid chromatography electrochemical detection (HPLC-ECD). Although this method is accurate, it requires a filter treatment process, which is time-consuming and costly for an environmental monitoring tool, in which many samples are measured simultaneously. Therefore, in this study, we investigated a simple immunoblotting method in which atmospheric PM proteins were directly transferred to a polyvinylidene fluoride (PVDF) membrane and measured using an anti-3-NT antibody (the filter blot method). The 3-NT value obtained from this method was significantly correlated (r = 0.809, p < 0.001) with that of the HPLC-ECD method, with a detection power of 0.1 μg/mL for tyrosine nitrated bovine serum albumin equivalents. Multiple regression analysis using the filter blot method showed that the amount of 3-NT in atmospheric PM was significantly associated with the published environmental measurements of O₃ and PM in the region. Therefore, the filter blot method may be useful for the environmental monitoring of 3-NT in atmospheric PM.