Novel Chemical Tools and Methods for Quantitative Mass Spectrometry-Based Proteomics

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 increased samples and replicates to ensure statistical and analytical relevance. Towards increasing the throughput of CFTR quantitation, an ultrathroughput multiple reaction monitoring (uMRM) method was designed. CFTR digest samples, containing one common internal standard, were derivatized with unique mass tags. Derivatized, cysteinyl CFTR peptides were then enriched with an avidin/biotin pull-down strategy before MRM measurements. A 5-plex experiment was designed, and will be compared to traditional MRM measurements of the 5 samples analyzed individually. To evaluate a possible new mass tagging strategy for uMRM, the collisional fragmentation of peptides whose amine groups were derivatized with five linear _-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid, were investigated. Tandem mass spectra of the derivatized peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization.