We applied 1D-PAGE coupled with LC-MS/MS to the study of the airway epithelium proteome and its response to cigarette smoke exposure. This study presents the first proteomic profile of a relatively pure population of bronchial epithelial cells obtained from bronchoscopy brushings. We also used differences in the rate of protein detection between never and current smokers to identify candidates for proteins that vary in abundance in response to tobacco-smoke exposure. The effect of smoking on several of these proteins was confirmed by Western blot. We also found that for many candidates, smoking similarly affected expression of the mRNA transcripts that gave rise to these proteins. This was accomplished by measuring gene expression in the same samples that were profiled at the proteomic level and in an independent data set. The majority of proteins identified by LC-MS/MS had detectable levels of their corresponding transcript by microarray. Differing methodologies may account for the stronger relationship between protein and gene expression reported here relative to prior studies
Analysis of the proteome using 1D-PAGE coupled with LC-MS/MS resulted in the detection of 41 proteins for which expression of corresponding transcripts was not detected by microarray. Some of these failures to detect transcript expression could represent technical limitations of the microarray platform. However, we were intrigued that several of the proteins whose transcripts were not detected by microarray represent erythrocyte-specific proteins. This suggests that: 1) the airway epithelial samples collected for this study were likely contaminated with erythrocytes, and 2) that more generally, stable proteins may be detected by proteomic methods long after the mRNA which encodes for them has disappeared.
Using habitual smoking as a paradigm for inhalational exposures affecting airway epithelium, we have identified changes in protein among smokers by LC-MS/MS and validated select changes with Western blotting. A decrease in the short isoform of PLUNC has previously been described in the pooled nasal lavage fluid of current smokers when compared with nonsmokers
. Although the exact function of this protein is unclear, it is thought to act in the inflammatory response to inhaled irritants such as tobacco smoke. Other studies have demonstrated decreased levels of uteroglobin, an anti-inflammatory protein secreted by Clara cells, in the BAL
, pooled nasal lavage fluid
, and serum
of healthy smokers and in the bronchial epithelium of former smokers with COPD undergoing lung transplantation
. P4HB has been detected in a proteomic analysis of cell surface proteins of a lung adenocarcinoma cell line
and in the 2DE-proteomic analysis of resected lung adenocarcinomas
. This protein may function in the anti-oxidant response to cigarette smoke
. Other proteins with oxidoreductase activity identified by this approach, such as ALDH3B1, have not previously been linked to cigarette smoking at the protein level but may function in the airway epithelial response to the toxins in cigarette smoke. None of the proteins differentially detected in smokers in this study overlapped with proteins previously described as differentially expressed in the lungs of Winstar rats exposed to cigarette smoke
, or proteins differentially detected by 2DE/MALDI-TOF in a human pneumocyte cell line exposed to cigarette smoke extract
This study was limited by a relatively small sample size, the sensitivity of the proteomic technique, and challenges in the quantification of proteins. While age was a confounding variable in this study, the gene expression changes in the airway epithelium of never and current smokers were validated using age-matched samples from current and never smokers in a previously published gene-expression study 
, suggesting that the association between smoking-status and both gene and protein expression is unlikely to be due to differences in patient age. The amount of time elapsed between last smoking a cigarette and bronchoscopy was not recorded, and some of the variability of protein levels in Western blotting might relate to potential differences to the acute versus chronic effects of cigarette smoke. Although the small sample size limited the statistical analysis, Western blotting validated differences in protein detection identified by LC-MS/MS suggesting the method's potential specificity. However, the power of our study to detect additional proteomic changes that occurred in response to cigarette smoke exposure was limited. The sensitivity of this technology allowed detection of 859 proteins with a false positive rate of 1%. While this represents a small percentage of the total proteins present in epithelial cells, we have identified a greater number of proteins than previously used methods of sample collection and proteomic analysis for smokers and nonsmokers
. Because of the uncertainties associated with label-free quantification methods for the determination of protein expression levels, this platform serves mainly as a discovery tool. However, promising efforts in this area, including correlation of peak intensity or spectral counts with protein abundance, may soon remove this limitation
In summary, we have described the proteomic profile of normal bronchial epithelial cells using 1D-PAGE coupled with LC-MS/MS and linked this profile to smoking-induced transcriptional changes in these same cells. This approach has the potential to provide additional insight into host response to tobacco smoke and the pathogenesis of smoking-related lung disease.