Novel Chemical Tools and Methods for Quantitative Mass

Mass spectrometry-based proteomics utilizes a mass spectrometer to study the identity, quantity, localization, modification, interaction, and function of proteins. This technology was applied to quantify cystic fibrosis transmembrane conductance regulator (CFTR) protein, whose mutation is responsible for the lethal disease cystic fibrosis. Mutated CFTR is degraded before it reaches the plasma membrane (PM), where it performs its vital function as a chloride ion channel. The first step of drug modulation is to increase the expression of CFTR in the PM; thus, an accurate measurement of CFTR in the PM is desired. A tandem enrichment strategy of cell-surface biotinylation and gel electrophoretic enrichment, with pulse chase of stable isotopes, was applied to measure the lifetime of CFTR, in the apical PM of BHK-wtCFTR cells. The half-life was determined to be 29.0±2.5h. Quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate the understanding of the cystic fibrosis disease mechanism and thus the development of new disease-modifying drugs. CFTR and all proteomic quantitation suffers from low sample numbers and replicates due to lengthy analysis time. However, small changes in protein concentration demand increase