Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Food safety assessment in genetically engineered canola – LC-MS/MS as an alternative to antibody-based approaches (#73)

Michelle Colgrave 1 , Keren Byrne 1 , Joanne Caine 2 , Lukasz Kowalczyk 2 , Sapna Vibhakaran Pillai 3 , Bei Dong 3 , Susan MacIntosh 4 , Judith Scoble 2 , James Petrie 3 , Surinder Singh 3 , Xue-Rong Zhou 3
  1. Agriculture & Food, CSIRO, St Lucia, QLD, Australia
  2. Manufacturing, CSIRO, Parkville, VIC, Australia
  3. Agriculture & Food, CSIRO, Black Mountain, ACT, Australia
  4. NuSeed Americas, Alsip, IL, USA

Omega-3 oils are highly sought after for their human health benefits such as cardiovascular protection and improved cognitive development and function. Originally sourced from fish, growing concerns with food security, fish stocks, and industrial fishing have led to increased interest in alternative sources of omega-3 oils. Using gene technology, the ability to produce omega-3 oils from lower plants was transferred into canola, by introducing seven transgenes, representing the most complex piece of metabolic engineering so far achieved in plants.

For genetically modified products, food/feed risk assessment involves evaluation of protein stability and plant expression levels. Protein digestibility is considered a determinant of potential allergenicity that has been traditionally measured using antibodies. Yet membrane proteins are extremely difficult to express in heterologous systems making raising antibodies impracticable. In this study a novel mass spectrometric approach was developed that enabled the investigation of the in vitro digestibility of the seven transmembrane proteins. The proteins and their peptide fragments were identified by high resolution LC-MS/MS and subsequently LC-MRM-MS was employed for specific peptide quantitation.

A two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers spanning the length of each desaturase protein were monitored and showed that all regions were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes showing limited resistance to gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and ∆6-elongase revealed rapid protein hydrolysis and no fragments (>3 kDa) remaining after 60 minutes, complementing the novel approach. The LC-MS approach was sensitive, selective and did not require the use of purified proteins and is widely applicable to food/feed and environmental safety assessment.

LC-MS innovations in food safety assessment have facilitated the deregulation of a sustainable plant resource capable of producing omega-3 oils.