Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Applying proteomics to deliver superior and safe plant-based food products (#77)

Keren Byrne 1 , Michelle Colgrave 1 , Lukasz Kowalczyk 2 , Sapna Vibhakaran Pillai 3 , Bei Dong 3 , Geoff Dumsday 4 , Judith Scoble 2 , James Petrie 3 , Surinder Singh 3 , Sue Macintosh 5 , Xue-Rong Zhou 3
  1. CSIRO, Agriculture and Food, Brisbane, QLD, Australia
  2. CSIRO, Agriculture and Food, Parkville, VIC, Australia
  3. CSIRO, Agriculture and Food, Black Mountain, ACT, Australia
  4. CSIRO, Agriculture and Food, Clayton, VIC, Australia
  5. Nuseed Americas, Illinois, USA

The omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA), eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid (EPA-20:5ω3; DPA-22∶5ω3; DHA-22∶6ω3) are widely recognised as beneficial to human health. Through metabolic engineering seven fatty acid desaturases and elongases were introduced into canola to convert oleic acid (OA) to DHA in a single pathway expression vector. For genetically modified products, food/feed and environmental risk assessment is required and involves evaluation of each transgenic protein, including protein stability and plant expression levels. Mass spectrometry-based proteomics was employed to demonstrate the gastrointestinal stability of the seven ω3LCPUFA enzymes. Current antibodies, have not proved useful in the characterization of these enzymes because of tight membrane association and non-specific cross-reactions. Therefore, we developed LC-MS/MS based methods to evaluate these transgenic membrane proteins. The proteins were digested with pepsin under simulated gastric fluid conditions for up to 60 min, followed by complete trypsin digestion. The decline of tryptic peptides was used as a proxy for intact protein, and the appearance of peptic peptides indicated the in vitro digestibility of the transgenic proteins. We applied a similar principle to quantify each target protein in different plant tissues by LC-MS/MS. The level of tryptic peptide markers, in 250 µg of total protein, was quantified using a spiked internal standard. The results from an example study demonstrated that >80% of the full-length protein was digested within 10 or >93% after 60 min of incubation. Applying the LC-MS/MS method in transgenic plants; we demonstrate that seed-specific promoters correctly regulated expression of transgenes only in developing and mature seed, and that the enzymes were present at low levels (ng per mg). By examining specific peptides (unique to the targets), this approach provides highly selective and sensitive measurement of membrane proteins. The LC‑MS/MS methods described here are applicable to food/feed and environmental safety assessment.