Figure 3.

Bottom-up MS of mVDAC1 photolabeled with cholesterol analogues maps a cholesterol-binding site. A, representative MS1 doublet corresponding to the peptide GLTFTEKW labeled with KK174 (z = 4) and coupled to light and heavy FLI-tag. B, fragmentation (ETD) spectra corresponding to the heavy feature in A. The site defining ions localize KK174 labeling to Glu⁷³. C, a representative MS1 doublet corresponding to the peptide NTDNTLGTEITVEDQLARGL labeled with LKM38 (z = 4) and coupled to light and heavy FLI-tag. D, fragmentation (ETD) spectra corresponding to the light feature in C. Site defining ions localize LKM38 labeling to Thr⁸³. E, a cholesterol-binding pose from AutoDock bound by Tyr⁶², Phe⁷¹, Glu⁷³, Thr⁸³, Ile⁸⁵, and Ser¹⁰¹. F, same cholesterol binding pose as in E showing the aliphatic tail 3.5 Å from Glu⁷³ and the 7 position 2.5 Å from Thr⁸³, consistent with KK174 and LKM38 labeling of these residues.