Title

Targeted mass spectrometric quantification of apical plasma membrane cystic fibrosis transmembrane conductance regulator (CFTR)

Date of Completion

January 2009

Keywords

Chemistry, Analytical

Degree

Ph.D.

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ion channel in the apical plasma membrane of epithelial cells. Mutations in the gene coding for CFTR cause cystic fibrosis (CF). A major cellular disorder is insufficient apical plasma membrane localization of the protein. Its correction is important for developing CF therapeutics and treatments and requires a sensitive and precise method for quantifying apical plasma membrane CFTR. In this study, the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of CFTR tryptic peptides that were used to quantify endogenous and over-expressed CFTR protein in HT29 and MK cells. It was implemented by first selecting a surrogate marker peptide (designated as CFTR01, with amino acid sequence of NSILTETLHR) for analysis from a tryptic digest of purified CFTR protein. In addition, enrichment techniques via surface biotinylation and gel electrophoretic enrichment were used to reduce sample complexity and facilitate the MRM quantitation of endogenous CFTR. The enriched sample was digested in-gel from which the tryptic peptides were extracted, and a stable isotope internal standard peptide (CFTR01-18O 4) was added prior to mass spectrometric analysis. The absolute amounts of CFTR from cell samples were inferred from the relative signal intensities of the ion chromatograms generated from the MRM transitions of CFTR01 and CFTR01-18O4. Using this analytical strategy, endogenous and overexpressed CFTR from HT29 and BHK cells were quantified. CFTR quantified using this method showed 30 times improvement in the sensitivity of the analysis of overexpressed apical plasma membrane CFTR over in-solution digested samples that were surface biotinylated. ^