Shotgun Proteomic Analysis of Yeast-Elicited California Poppy (Eschscholzia californica) Suspension Cultures Producing Enhanced Levels of Benzophenanthridine Alkaloids

The California poppy, Eschscholzia califomica, produces benzophenanthridine alkaloids (BPAs), an important class of biologically active compounds. Cell cultures of E. californica were investigated as an alternative and scalable method for producing these valuable compounds; treatment with yeast extract increased production from low levels to 23 mg/g dry weight (DW) of BPAs. A shotgun proteomic analysis of E. califomica cell cultures was undertaken to explore changes in metabolism associated with enhanced BRA production. We implemented differential centrifugation and then shotgun proteomics based on nanoliquid chromatography/mass spectrometry (nano-LC-MS/MS) for peptide separation and analysis. A unigene database available for E. califomica was translated and utilized for protein identification. Approximately 646 proteins (3% false discovery rate at the protein level) were identified. Differentially abundant proteins observed with elicitation included enzymes involved in (S)adenosyl methionine (SAM) biosynthesis and BRA biosynthesis. These results demonstrate (1) the identification of proteins from a medicinal plant using shotgun proteomics combined with a well-annotated, translated unigene database and (2) the potential utility of proteomics for exploring changes in metabolism associated with enhanced secondary metabolite production.