Analysis of unintended open reading frame ORF-130 expression in maize event MZIR098 by LC-MS as part of the allergenicity risk assessment of genetically modified crops

Rigorous safety assessments are required for genetically modified (GM) crops to support their use as food or feed. These assessments include bioinformatics analysis of the amino acid sequences of all potential open reading frames (ORFs) encoded in the exogenous DNA introduced into the plant through genetic modification. Sequence similarities of potential DNA products to known or potential toxins and allergens trigger subsequent assessments of potential hazards for the consumer or the environment. The bioinformatic analysis of Syngenta maize event MZIR098 insert DNA revealed a total of 415 ORFs. One putative ORF (ORF-130) triggered further risk assessment based on its sequence similarities with known allergens, including peanut allergen Ara h 1, wheat glutenin, and others. We examined the potential expression of ORF-130 at a protein level to exclude the possibility of exposure during consumption of MZIR098 maize. Specifically, we developed and validated a liquid chromatography approach coupled to a tandem mass spectrometry (LC-MS/MS) method, called parallel reaction monitoring (PRM). This measure of expression was quantitative and targeted against the peptides unique to ORF-130. The linear range of the method was 25-5000 amol ORF-130/µl. Both, limit of detection (LOD) and lower limit of quantitation (LLOQ) of the method, were 12 μg ORF-130 per gram of dry MZIR098 maize leaf. The method was accurate and specific to ORF-130. PRM analysis of MZIR098 extracts did not detect putative ORF-130 above the limits of detection/quantitation. Furthermore, the broad proteomic data dependent acquisition (DDA) analysis of the MZIR098 extracts demonstrated no evidence of ORF-130 corroborating the PRM analysis. Our results confirm that ORF-130 is not characterized as a functional gene in Syngenta maize event MZIR098 insert DNA and does not express gene products at detectable levels.