Multiplexed protein quantification methods for plant tissue are becoming increasingly important with the development of genetically modified crops that express multiple transgenes or �stacked traits�.  The current industry standard for quantification of transgenic proteins is enzyme linked-immunosorbent assay (ELISA), which typically quantifies one protein at a time. An alternative quantitative method to ELISA is liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) combined with selected reaction monitoring/multiple reaction monitoring (SRM/MRM). We have compared validated ELISA methods with an LC-MS/MS/SRM method, for multiplexed protein quantification, using a commercial stacked trait crop that expresses six transgenic proteins: phosphinothricin-N-acetyltransferase (PAT), phosphomannose isomerase (PMI), vegetative insecticidal protein 3A (VIP3A), modified Cry3A (mCry3A), mutated 5-enol pyruvylshikimate-3-phosphate synthase (mEPSPS), and Cry1Ab.   The LC-MS/MS/SRM method delivered results comparable to ELISA results and additionally, was able to differentiate and selectively quantify both an endogenous 5-enol pyruvylshikimate-3-phosphate synthase and a mutated version of 5-enol pyruvylshikimate-3-phosphate synthase protein that differs by only two amino acids.

Comparison of LC-MS/MS/SRM to ELISA for multiplexed absolute quantification of genetically modified traits in maize leaves

248th National Meeting (2014)

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