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

Electrophoresis. 2005 Jun;26(11):2092-108. doi: 10.1002/elps.200410428.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Air Pollutants / adverse effects
  • Cell Line
  • Electrophoresis, Gel, Two-Dimensional
  • Environmental Exposure / adverse effects
  • Epithelial Cells / metabolism*
  • Fluorescent Dyes
  • Humans
  • Macrophages / metabolism*
  • Oxidative Stress*
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism*
  • Signal Transduction*
  • Vehicle Emissions / toxicity*

Substances

  • Air Pollutants
  • Fluorescent Dyes
  • Phosphoproteins
  • Vehicle Emissions