Quantification of intractable membrane proteins in genetically engineered crops by liquid chromatography coupled with tandem mass spectrometry
Liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods to quantify a membrane protein in genetically engineered maize leaves and another in soybean seeds were developed and validated. An extraction buffer containing 100 mM ammonium bicarbonate, 0.5% CHAPS (w/v) and 0.1% Triton X-100 (v/v) was determined to be suitable. High recovery (>88%) and good reproducibility (coefficient of variance ≤ 15%) were demonstrated for both membrane proteins. Recombinant proteins were used as standards to prepare calibration curves with good linearity (correlation coefficient > 0.997) and accuracy (deviation from nominal concentration <15%). Our study demonstrates the suitability of LC-MS/MS for quantification of intractable membrane proteins in genetically engineered crops.