title: De novo biosynthetic profiling of high abundance proteins in cystic fibrosis lung epithelial cells.
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.
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University School of Medicine, Bethesda, Maryland 20814, USA. hpollard@usuhs.mil
authors.forename: Harvey B
authors.initials: HB
authors.lastname: Pollard
authors.forename: Ofer
authors.initials: O
authors.lastname: Eidelman
authors.forename: Catherine
authors.initials: C
authors.lastname: Jozwik
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Meera
authors.initials: M
authors.lastname: Srivastava
authors.forename: Xia D
authors.initials: XD
authors.lastname: Ji
authors.forename: Brighid
authors.initials: B
authors.lastname: McGowan
authors.forename: Christine Formas
authors.initials: CF
authors.lastname: Norris
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Todo
authors.forename: Thomas
authors.initials: T
authors.lastname: Darling
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Mogayzel
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Zeitlin
authors.forename: Jerry
authors.initials: J
authors.lastname: Wright
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.forename: Eleanore
authors.initials: E
authors.lastname: Metcalf
authors.forename: William J
authors.initials: WJ
authors.lastname: Driscoll
authors.forename: Greg
authors.initials: G
authors.lastname: Mueller
authors.forename: Cloud
authors.initials: C
authors.lastname: Paweletz
authors.forename: David M
authors.initials: DM
authors.lastname: Jacobowitz
mesh_terms: D002460:Cell Line; D003550:Cystic Fibrosis / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis*; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013462:Sulfur Radioisotopes
chemical_list: D020543:Proteome; D013462:Sulfur Radioisotopes; D008715:Methionine