Figure 7
Discovery of active P450s from the fungal plant pathogen Zymoseptoria tritici. (a) Enrichment of Zymoseptoria tritici P450s upon labeling with DB096. Microsomes isolated from Z. tritici cell cultures were incubated with 30 μM DB096 or an equal volume of DMSO in the presence of 1 mM NADPH for 1 h. Labelled proteins were subjected to click chemistry to attach biotin, enriched via streptavidin pull-down, and on-bead-digests (OBD) were analysed by LC–MS/MS. Volcano plots highlight proteins depleted and enriched by DB096 labelling of Z. tritici microsomes labelling. Enrichment is represented by the log2FC (P450 probe/CC) against the significance (− log10FDR) between the click control (CC) and DB096 using replicates. The dots indicate whether proteins were significantly enriched (black lines) or depleted (grey lines) in the DB096 sample. (b) Many P450s are detected in in-solution-digests (ISD) Z. tritici microsomes. Z. tritici microsomes were digested with Lys-C and trypsin and digested peptides were analysed by LC–MS/MS. All identified proteins were ranked by their spectral intensity and plotted against the spectral intensity. Lines indicate P450s that were enriched upon DB096 labeling (solid lines) or not enriched (dashed lines) or not detected (striped lines). (c) List of P450s detected by ISD in (b). Shown are the rank position in MS intensity, the UniProt accession code, the P450 name, the P450 name given by Donzelli, the genomic accession code and the detection of the protein upon on-bead digest (a), given with P-value if enriched, non-significant (ns) or not detected (–).
