Figure 5
Labeling of P450s in mouse liver microsomes. (a) NADPH-dependent labeling in mouse liver microsomes. Microsomes were labeled for 1.5 h with 20 μM probe with and without 1 mM NADPH. Labeled proteins were coupled to Cy5-picolyl-azide via click chemistry, separated on SDS-PAGE gels and visualized by in-gel fluorescence scanning. (b) Distribution graph summarizing proteins identified upon purification of labeled proteins from mouse liver microsomes. Microsomes were incubated for 1.5 h with 30 μM DB096 or an equal volume of DMSO in the presence of 1 mM NADPH. Both samples were coupled to azide-biotin via click chemistry, and proteins were purified on streptavidin beads, digested with trypsin (on-bead-digest, OBD), and analyzed by MS. The total MS signal intensity (y-axis) was plotted against the distribution (x-axis), which was calculated as the ratio between spectral intensities of the DB069 sample divided by the sum of DB096 and the click control (CC) sample (DB069/(DB069 + CC)) for n = 1 replicate. Highlighted are all P450 enzymes (red) and other proteins (grey). (c) Shotgun proteomics of the input sample. Mouse liver microsomes were digested with trypsin (in-solution-digest, ISD) and analyzed by MS. Proteins were ranked on their total MS spectral intensity and the P450s are highlighted (red) with those detected and enriched in (b) show as solid lines. *Detected by on-bead digest in (b).
