Isolation of rodent airway epithelial cell proteins facilitates in vivo proteomics studies of lung toxicity.
Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered 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 that facilitates in vivo proteomics studies of two target-cell pheno-types 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 fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.
Wheelock
Asa M
AM
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. amkarlsson@ucdavis.edu
Zhang
Lu
L
Tran
Mai-Uyen
MU
Morin
Dexter
D
Penn
Sharron
S
Buckpitt
Alan R
AR
Plopper
Charles G
CG
D000199:Actins / Q000032:analysis; D000818:Animals; D018893:Bronchoalveolar Lavage; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000737:chemistry* / Q000648:ultrastructure; D015151:Immunoblotting; D008297:Male; D008689:Methacrylates; D008855:Microscopy, Electron, Scanning; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020545:Respiratory Mucosa / Q000737:chemistry* / Q000166:cytology
D000199:Actins; D008689:Methacrylates; D003577:Cytochrome P-450 Enzyme System