Project Summary Cystic Fibrosis (CF) is caused by mutation of the CFTR gene and is one of the most common inherited childhood diseases, impacting 1 in 4,000 children born in the US (www.cff.org). Today, CF disease symptoms in patients with the most common mutation (∆F508 CFTR) can be improved with three different drug combinations. However, the drugs have negative side effects that reduce patient compliance to therapeutic regimens and pose long-term health risks. Additionally, many other common CF-causing mutations respond poorly or not at all to any of the current CF drugs, leaving CF patients carrying these mutations only with symptomatic therapy. Efforts to develop new compounds for such CF variants are hampered by the lack of protein structures that would reveal the conformational defects of these variants, mostly due to technical difficulties in expressing and purifying sufficient quantities of these unstable proteins. To characterize the conformational defects of misfolded CFTR variants and to aid in the development of new therapies, we previously developed Covalent Protein Painting (CPP), a novel method for in vivo structural characterization of proteins by mass spectrometry. Here, we propose to develop a more sensitive and multiplexable CPP method, named bioTMT-CPP, that will facilitate detection and comparison of CFTR conformational changes between samples. The new method will facilitate the characterization of conformational defects in misfolded CFTR variants that are refractory to current therapies. Furthermore, our approach has the potential to pinpoint drug binding sites and identify the mechanism of action of current CF drugs, which remain unknown for three of the four active compounds. Such knowledge will help to rationalize drug combination therapies. We also propose to functionally characterize a novel CFTR conformation that we discovered by CPP and that is attained by misfolded and inactive CFTR, likely during protein trafficking. Insight into the molecular mechanisms that stabilize this conformation as well as those that release it into an active conformation will be invaluable for further corrector drug development and will benefit all CF patients.