De Novo Biosynthetic Profiling of High Abundance Proteins in Cystic Fibrosis Lung Epithelial Cells

Harvey B Pollard,Ofer Eidelman,Catherine Jozwik,Wei Huang,Meera Srivastava,Xia D Ji,Brighid Mcgowan,Christine Formas Norris,Tsuyoshi Todo,Thomas Darling,Peter J Mogayzel,Pamela L Zeitlin,Jerry Wright,William B Guggino,Eleanore Metcalf,William J Driscoll,Greg Mueller,Cloud Paweletz,David M Jacobowitz

doi:10.1074/mcp.m600091-mcp200

Abstract

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Highlights

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry
Using an approach of 2-D gel electrophoresis (2DGE) and mass spectrometry, we recently identified 194 unique high abundance proteins in the CF lung epithelial IB3-1 cell system [13]
Preliminary comparisons between the IB3-1 cells and IB3-1/S9 cells, a daughter cell line repaired by gene transfer with wild type CFTR, revealed that relatively few changes in silver-stained features could be detected for any of the 194 high abundance proteins

Summary

Introduction

In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry Using an approach of 2-D gel electrophoresis (2DGE) and mass spectrometry, we recently identified 194 unique high abundance proteins in the CF lung epithelial IB3-1 cell system [13]. Preliminary comparisons between the IB3-1 cells and IB3-1/S9 cells, a daughter cell line repaired by gene transfer with wild type CFTR, revealed that relatively few changes in silver-stained features could be detected for any of the 194 high abundance proteins.

Results

Conclusion