Use of a fluorescent phosphoprotein dye to characterize oxidative stress-induced signaling pathway components in macrophage and epithelial cultures exposed to diesel exhaust particle chemicals

A large body of evidence has shown that exposure to ambient particulate matter (PM) leads to asthma exacerbation through an excitation of allergic inflammation. Utilizing diesel exhaust particles (DEPs) as a model air pollutant, we and others have demonstrated that PM contains redox-active chemicals that generate inflammation through an oxidative stress mechanism. Recently, the strengths of proteomics have enabled us to demonstrate that organic DEP extracts induce a hierarchical expression pattern of oxidative stress-induced proteins in macrophages and epithelial cells. As a further extension of this work, we now employ a new phosphosensor fluorescent dye, Pro-Q Diamond, to elucidate the induction of phosphoproteins and intracellular signaling cascades that may play a role in DEP-induced inflammation. We demonstrate that DEPs induced the phosphorylation of several phosphoproteins that belong to a number of signaling pathways as well as other oxidative stress pathways. In combination with cytokine array, phosphoproteome analysis using Pro-Q Diamond allowed us to characterize the aromatic and polar chemicals of DEPs that are involved in the activation of three different mitogen-activated protein (MAP) kinase signaling pathways.