Direct detection of 3-nitrotyrosine reveals the nitration of proteins in laboratory exposure and ambient aerosols

Tyrosine residues in proteins can be nitrated to form 3-nitrotyrosine (3-NT) under the influence of ozone (O₃) and nitrogen dioxide (NO₂) in the air, which may introduce health impacts. A selective and sensitive enzyme-linked-immunoassay (ELISA) method was developed to determine 3-NT in modified model protein (bovine serum albumin, BSA) and ambient aerosol samples. The nitration degrees (NDs) of BSA in the exposure experiments with different durations were detected by both the ELISA and spectrophotometric methods (i.e., ND_ELISA and ND_SEC-PDA), which show good coincidence. The kinetic investigation by both ΔND_ELISA and ΔND_SEC-PDA in the exposure experiments shows that the rate coefficients (k) of the pseudo-first-order kinetic rate reactions of protein nitration were comparable. These results indicate that direct detection of 3-NT by the ELISA method can be applied for laboratory exposure samples analysis for kinetic studies. Based on the selective detection of 3-NT, ND_ELISA provides a promising measure for the assessment of ND in model proteins. 3-NT was also measured in PM_2.5 samples in summer in Guangzhou, southern China, ranging from 10.1 to 404 pg/m³, providing clear evidence of protein nitration in ambient aerosols. We further proposed that 3-NT/protein can be used as a proxy to evaluate protein nitration in ambient aerosols. A significant correlation was observed between 3-NT/protein and O₃, confirming the crucial role of O₃ in protein nitration. Our results show that the direct detection of 3-NT by the ELISA method can be more widely applied in the laboratory and field-based studies for understanding the mechanisms of protein nitration.