Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational...

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.