Using 2D-PAGE And iTRAQ Proteomics Approaches To Investigate The Effect Of Cigarette Smoke Fraction Exposure On Primary Normal Human Bronchial Epithelial (NHBE) Cells
Presented at HUPO 2013
In order to update and advance our knowledge regarding the impact of cigarette smoke on the human bronchial epithelium, a systems biology approach that combines state-of-the-art proteomics, together with gene expression analysis and a toxicological endpoint, was developed. Primary normal human bronchial epithelial (NHBE) cells derived from a healthy non-smoker adult donor were used and were exposed for 24 hours to a solution of phosphate buffered saline through which smoke from the reference cigarette 3R4F had been bubbled (smoke bubbled PBS, SBPBS). The dose applied was selected based on survival assay results showing less than 20% cytotoxicity after 24 hours of exposure. Protein lysates obtained from NHBE cells exposed to SBPBs, and control exposed only to SPBS, were analyzed using 2D-PAGE and iTRAQ (isobaric tags for relative and absolute quantitation) proteomics approaches to detect differentially expressed proteins. Three technical replicates were analyzed to assess repeatability and reproducibility across measurements. Analyses for the verification and quantification of selected targets, identified from the 2D-PAGE and iTRAQ proteomics approaches, using either western blots or selected reaction monitoring-like approach (SRM-like), were performed. The generated proteomic datasets of differential proteins and the selected verified ones will be presented. Those results will complement the analyses performed on the other endpoints (gene expression and transcriptomics) captured within this study and will further establish the foundation of PMI’s systems biology approach to assess the impact of modified risk tobacco products (MRTP).