20-107 Sigma-AldrichPeroxynitrite

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are derived as a result of inflammation and oxidative stress and can result in protein modifications. As such, these protein modifications are used as biomarkers for inflammation and oxidative stress. In addition, modifications in single-tissue-associated proteins released into blood can provide insight into the tissue localization of the inflammation or oxidative stress. We have developed an enzyme-linked immunosorbent assay antibody microarray platform to analyze the levels of 3-nitrotyrosine in specific proteins in a variety of biological samples, including human plasma and sputum. Selective-capture antibodies are used to immunoprecipitate individual proteins from samples onto isolated spots on the microarray chips. Then, a monoclonal antibody for 3-nitrotyrosine is used to detect the amount of 3-nitrotyrosine on each spot. Our studies suggest that this approach can be used to detect trace amounts of 3-nitrotyrosine in human plasma and sputum. In this paper, we describe our antibody microarray protocol for detecting 3-nitrotyrosine in specific proteins.

Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.