Agricultural biotechnology relating to crop protection has evolved toward increasing numbers of transgenic proteins being co-expressed or �stacked� to achieve tolerance to multiple herbicides or multiple modes of action to insect resistance. Stacked proteins present a challenge to traditional technologies for quantifying transgenic protein expression such as Enzyme Linked Immunosorbent Assays (ELISA) to accommodate the volume of data generated per sample. A selective and sensitive multiplex method was developed and validated to quantify the double mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS), aryloxyalkanoate dioxygenase (AAD-12), and phosphinothricin acetyltransferase (PAT) proteins in soybean tissues using tandem mass spectrometry (LC-MS/MS). Signature peptides representing each protein were identified by an information-dependent acquisitions (IDA) followed by optimization in multiple reaction monitoring (MRM) for quantitation with a hybrid Q-TRAP mass spectrometer. The method was validated for its accuracy, precision, linearity, and limits of detection and quantitation in various soybean tissues as well as signature peptide stability. Whole protein calibration curves were compared to synthetic signature peptide calibration curves during the validation with heavy labeled synthetic peptides serving as internal standards. Finally, the validated LC-MS/MS method was compared to validated ELISA methods by examining protein expression across three generations of transgenic soybean root, grain, and leaf tissues. The stacked event that was analyzed in this abstract was co-developed by Dow AgroSciences and MS Tech.

Multiplexing protein quantitation of transgenic proteins in soybean tissues using tandem mass spectrometry (LC-MS/MS)

246th National Meeting (2013)

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