P109 Label-Free Quantitation of Chorismate Biosynthesis Pathway Protein Expression in Yeast Following Treatment With Glyphosate

Christopher A Bolcato , Protea Biosciences, Morgantown, WV
Jennifer A Gallagher , West Virginia University, Morgantown, WV
Gregory Boyce , Protea Biosciences, Morgantown, WV
Callee Walsh , Protea Biosciences, Morgantown, WV
Matthew D Maust , Protea Biosciences, Morgantown, WV
Sujatha Chilakala , Protea Biosciences, Morgantown, WV
The wide spread use of the herbicide glyphosate can be found on many different genetically modified crop plants. Glyphosate’s mode of action is to block the activity of the enzyme 5-Enolpuruvylshikimate 3-phosphate synthase (EPSP synthase). This eventually leads to the death of the organism due to lack of aromatic amino acid production. This pathway is very specific to plants and microorganisms and therefore, toxicity in humans is thought to be minimal. Acute toxicity in humans has not been observed, however the chronic effects are not yet understood. In this work, we used a nanoLC-MS/MS based, global proteomics and label-free quantitation approach for investigation of potential pathways effected by glyphosate exposure in different strains of Saccharaomyces cerevisiae. Four different strains of Saccharomyces cerevisiae (RM11a, AWRI1631, GSY147 and YJM789K5a) with varying levels of glyphosate resistance were grown to mid log phase in yeast minimal media (YM) to a 0.25 OD600, split and grown for 90 minutes with and without 0.25% glyphosate treatment. Cells were lysed and digested with trypsin for proteomics analysis. Sample analysis was conducted using nanoLC and a Q Exactive Orbitrap operating in positive ion mode. Data Independent acquisition mode was used for MSMS data acquisition with the top 10 ions selected for MSMS. Proteomics LC-MS/MS data was processed using Proteome Discoverer (V 2.1, Thermo). Search engines included Sequest HT and Mascot. Fold-changes in protein expression were determined across control and glyphosate-treated samples. Using global proteomics and label-free relative quantitation, differential fold-change analysis was performed on over 1100 proteins identified from 400ng of lysate. Chorismate pathway proteins such as ARO1, ARO3, TRP2 and TRP5 were determined to be affected by glyphosate treatment.