Figure 4
Maize ZmCYP81A9 and N. benthamiana P450s are labeled with P450 probes. (a) Volcano plots for detection of labeled proteins purified from microsomes of ZmCYP81A9-GFP-expressing leaves incubated with DB088 (left) or DB096 (right). Microsomes isolated from agroinfiltrated leaves expressing ZmCYP81A9-GFP were labeled with and without 10 μM probes in the presence of 1 mM NADPH for 1.5 h. Labeled proteins were coupled to azide-biotin using click chemistry and biotinylated proteins were purified on streptavidin beads, digested with trypsin (on-bead-digest, OBD) and released peptides were identified by MS. The log2 fold change (log2FC) was plotted against the significance (− log10FDR) for n = 3 biological replicates. Highlighted are proteins that are significantly more (green) or less (red) abundant in the probe labeled samples when compared to the click-chemistry control, showing P450s as diamonds. (b) Shotgun proteomics of the input sample. Microsomes isolated from agroinfiltrated leaves expressing ZmCYP81A9-GFP 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. Identified P450s and their rank in the ISD dataset and the log2 of the MS intensity, followed by the adjusted P-values in on-bead digest analysis presented in (a). *The annotation of N. benthamiana P450s is based on the closest homology.
