Anthropogenic air pollutants are known to be a significant factor
in causing lung disease, which is a leading cause of morbidity and mortality in urban areas worldwide. Great efforts have been made to elucidate the mechanisms of toxicity of various air pollutants, but most testing performed to date has been focused on the effects of a single toxicant. This
approach of single-toxicant testing mainly results from limitations
in techniques available to study the complex effects of exposure
to multiple chemicals.
Recent developments in genomics, proteomics and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell-phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins, which facilitates in vivo proteomics studies of two target-cell phenotypes of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The
method yields epithelial cell-specific proteins in 5-fold higher concentrations
and reduces the yield of non-epithelial cell proteins 13-fold in comparison to
samples from microdissected airways.
In spite of the well-publicized problems inherent in the separation and measurement of protein abundance by 2-dimensional electrophoresis (2DE), this technique remains the most capable method for separation of complex proteomes. Two of the most critical issues that need to be addressed to improve the application of 2DE to proteomic studies are inter-gel variation and quantification. To decrease the effects of inter-gel variability, we have developed a fluorescent internal protein standard for use in 2DE analysis. Use of commercially available post-electrophoretic fluorescent protein stains for quantification yields a method with a significant dynamic range (104), which avoids problems associated with dual spot migration patterns observed in the well established DIGE method. We
are currently using these two newly developed methods to probe the underlying
mechanisms for the synergistic effects of two abundant air pollutants, ozone
and 1-nitronaphthalene, on injury to rat airway epithelial cells.