VX-770 probe photolabels full length Wt-CFTR protein on peptide (1049-1060) in MSD2
(A) After trypsin digestion of photolabeled CFTR, mass spectrometry studies were conducted to detect labeled residues. We show the spectra corresponding to the photolabeled peptide: 1049-1060. Left panel, MS1 spectrum of the 3 + precursor, monoisotopic mass is highlighted in green, m/z isolation window is highlighted in yellow. Right panel, MS2 fragmentation spectrum, diagnostic VX-770 reporter fragment ions are highlighted in orange.
(B) In complementary studies, we assessed photolabeling of independent domains and we show the boundaries of these CFTR fragments.
(C) Immunoblots of steady-state expression of WT-CFTR photolabeled with VX-770-Biot (0.1 μM) +/− VX-770 (10 μM) or SE-03 (10 μM). Bar graphs show the mean (±S.E.M) of the ratio of CFTR bound/CFTR unbound (n = 3-5). CFTR bound: CFTR biotinylated; CFTR unbound: CFTR unbiotinylated. (B) (∗p < 0.05, ∗∗p < 0.01)