Proteomics reveals the in vitro protein digestibility of seven transmembrane enzymes from the docosahexaenoic acid biosynthesis pathway. (August 2019)

• Record Type: Journal Article
• Authors: Colgrave, Michelle L.; Byrne, Keren; Caine, Joanne; Kowalczyk, Lukasz; Vibhakaran Pillai, Sapna; Dong, Bei; Lovrecz, George; MacIntosh, Susan; Scoble, Judith A.; Petrie, James R.; Singh, Surinder; Zhou, Xue-Rong
• Is Part Of: Food and chemical toxicology. Volume 130(2019)
• Page Start: 89
• Page End: 98
• Publication Date: 2019-08
• Subjects: Protein digestibility -- Genetic modification -- Omega-3 long-chain fatty acids -- Docosahexaenoic acid (DHA) -- Canola; Toxicology -- Periodicals; Food poisoning -- Periodicals; Food Poisoning -- Periodicals; Toxicology -- Periodicals; Toxicologie -- Périodiques; Intoxications alimentaires -- Périodiques; Food poisoning; Toxicology; Periodicals; Electronic journals; 615.9
• DOI: 10.1016/j.fct.2019.05.015
• Languages: English
• ISSNs: 0278-6915

Abstract

Abstract: The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro . Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3 kDa) remained after 60 min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins. Graphical abstract: Image 1 Highlights: Seven transmembrane enzymes were expressed in canola. A model for simulating gastric digestion was developed. Targeted LC-MS/MS analysis allowed measurement of in vitro digestion. The desaturase enzymes were readily degraded (>95% within 5 min). The novel LC-MS/MS methods were complemented by tradition gel-based analyses.