Using LC-MS Based Methods for Testing the Digestibility of a Non-Purified Transgenic Membrane Protein in Simulated Gastric Fluid

Skinner et al., J Agric Food Chem. (2016) - PMID: 27255301

Product(s) used in this publication:  Absolutely Quantified Peptides SpikeTides™ TQL

Abstract:

The digestibility of a nonpurified transgenic membrane protein was determined in pepsin, as part of the food safety evaluation of its resistance to digestion and allergenic potential. Delta-6-desaturase from Saprolegnia diclina, a transmembrane protein expressed in safflower for the production of gamma linolenic acid in the seed, could not be obtained in a pure, native form as normally required for this assay. As a novel approach, the endoplasmic reticulum isolated from immature seeds was digested in simulated gastric fluid (SGF) and the degradation of delta-6-desaturase was selectively followed by SDS-PAGE and targeted LC-MS/MS quantification using stable isotope-labeled peptides as internal standards. The digestion of delta-6-desaturase by SGF was shown to be both rapid and complete. Less than 10% of the initial amount of D6D remained intact after 30 s, and no fragments large enough (>3 kDa) to elicit a type I allergenic response remained after 60 min.

KEYWORDS:

LC-MS; food safety evaluation; genetically engineered plants; mass spectrometry; membrane proteins; protein digestibility

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