Shotgun Proteomic Analysis of Yeast-Elicited California Poppy (Eschscholzia californica) Suspension Cultures Producing Enhanced Levels of Benzophenanthridine Alkaloids John T. Oldham, † Marina Hincapie, ‡ Tomas Rejtar, ‡ P. Kerr Wall, § John E. Carlson, |,# and Carolyn W. T. Lee-Parsons* ,† Department of Chemical Engineering, 342 Snell Engineering Center, 360 Huntington Avenue, Northeastern University, Boston, Massachusetts 02115, Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, Massachusetts 02115, Department of Biology and The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, School of Forest Resources and The Huck Institutes of the Life Sciences, 323 Forest Resources Building, Pennsylvania State University, University Park, Pennsylvania 16802, and Department of Bioenergy Science and Technology (WCU), Chonnam National University, 333 Yongbongro, Buk-gu, Gwangju 500-757, Korea Received January 17, 2010 The California poppy, Eschscholzia californica, 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. californica cell cultures was undertaken to explore changes in metabolism associated with enhanced BPA 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. californica 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 BPA 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. Keywords: Eschscholzia californica • EST database • LC-MS/MS • secondary metabolism • shotgun proteomics Introduction The California poppy, Eschscholzia californica, produces benzophenanthridine alkaloids (BPAs) such as the biologically active compounds sanguinarine and chelerythrine. 1–6 Sangui- narine and chelerythrine (Figure 1) show activity against multiple targets associated with inflammation, 3 cell cycle regulation, and apoptosis of cancer cells. 4,6,7 In addition, sanguinarine was previously incorporated as an antiplaque agent in Viadent toothpastes and mouthwashes. 2 Because of BPA’s important biological activities, cell cultures of E. cali- fornica are being investigated as an alternative and scalable method for producing these valuable compounds. The advan- tage of the cell culture system is that environmental conditions can be controlled and easily manipulated for improving production. For example, BPA production is significantly enhanced in cell cultures of E. californica and Papaver somniferum with elicitation. 8–13 Elicitors activate plant natural defense responses, including increased secondary metabolite production. 14,15 Examples of elicitors include hormones (e.g., jasmonates or salicylic acid), crude biological extracts (e.g., fungal homoge- nate), and purified biological extracts (e.g., polysaccharides derived from the yeast cell wall). 16,17 In E. californica cell cultures, yeast elicitor is believed to increase BPA production by activating a pH-dependent signal transduction pathway. 18,19 Activation of signal transduction pathways triggers gene tran- scription leading to increased enzyme abundance and second- ary metabolite production. For instance, gene transcripts of several BPA biosynthetic enzymes were elevated by elicitation with yeast extract. 20–22 Also, the levels of 6 BPA enzymes were * To whom correspondence should be addressed. Phone: 617-373-2989. Fax: 617-373-2209. E-mail: clee@coe.neu.edu. † Department of Chemical Engineering, Northeastern University. ‡ Barnett Institute of Chemical and Biological Analysis, Northeastern University. § Department of Biology and The Huck Institutes of the Life Sciences, Pennsylvania State University. | School of Forest Resources and The Huck Institutes of the Life Sciences, Pennsylvania State University. # Department of Bioenergy Science and Technology (WCU), Chonnam National University. 10.1021/pr1000412  2010 American Chemical Society Journal of Proteome Research 2010, 9, 4337–4345 4337 Published on Web 08/06/2010