title: Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry.
abstract: Currently, traditional immunochemistry technologies such as enzyme-linked immunosorbent assays (ELISA) are the predominant analytical tool used to measure levels of recombinant proteins expressed in genetically engineered (GE) plants. Recent advances in agricultural biotechnology have created a need to develop methods capable of selectively detecting and quantifying multiple proteins in complex matrices because of increasing numbers of transgenic proteins being coexpressed or "stacked" to achieve tolerance to multiple herbicides or to provide multiple modes of action for insect control. A multiplexing analytical method utilizing liquid chromatography with tandem mass spectrometry (LC-MS/MS) has been developed and validated to quantify three herbicide-tolerant proteins in soybean tissues: aryloxyalkanoate dioxygenase (AAD-12), 5-enol-pyruvylshikimate-3-phosphate synthase (2mEPSPS), and phosphinothricin acetyltransferase (PAT). Results from the validation showed high recovery and precision over multiple analysts and laboratories. Results from this method were comparable to those obtained with ELISA with respect to protein quantitation, and the described method was demonstrated to be suitable for multiplex quantitation of transgenic proteins in GE crops.
authors.affiliation: Dow AgroSciences, LLC , 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Hill
authors.affiliation: Dow AgroSciences, LLC , 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
authors.forename: Trent J
authors.initials: TJ
authors.lastname: Oman
authors.affiliation: Dow AgroSciences, LLC , 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
authors.forename: Guomin
authors.initials: G
authors.lastname: Shan
authors.affiliation: Dow AgroSciences, LLC , 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
authors.forename: Barry
authors.initials: B
authors.lastname: Schafer
authors.affiliation: Critical Path Services, LLC , 3070 McCann Farm Drive, Garnet Valley, Pennsylvania 19060, United States.
authors.forename: Julie
authors.initials: J
authors.lastname: Eble
authors.affiliation: Critical Path Services, LLC , 3070 McCann Farm Drive, Garnet Valley, Pennsylvania 19060, United States.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Chen
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000123:Acetyltransferases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D005591:Chemical Fractionation; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D049308:Dioxygenases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D055550:Protein Stability; D011994:Recombinant Proteins / Q000032:analysis* / Q000378:metabolism; D012015:Reference Standards; D013025:Glycine max / Q000235:genetics*; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D011994:Recombinant Proteins; D049308:Dioxygenases; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase
keywords: ELISA; GE crops; LC-MS/MS; biotechnology; digestion efficiency; multiplex; protein quantitation; recombinant protein; stacked-trait product; surrogate peptide; validation