Analysis of isoprenyl-phosphates by liquid chromatography-mass spectrometry.

ABSTRACT

Isoprenoids in plants are synthesized following the plastidial methylerythritol-4-phosphate (MEP) pathway or the mevalonate pathway localized to the cytosol and peroxisomes. Isoprenyl-diphosphates (isoprenyl-PP) are important intermediates for the synthesis of chlorophyll, carotenoids, sterols, and other isoprenoids in plants. The quantification of isoprenyl-PP is challenging due to the amphipathic structure, the low abundance, and the susceptibility to hydrolysis during extraction and storage. Different methods for the measurement of isoprenyl-phosphates have been developed. Isoprenyl-phosphates can be measured after radioactive labeling or after derivatization. Liquid chromatography-mass spectrometry (LC-MS) methods provide enhanced sensitivity, but still require the extraction from large amounts of sample material. In the protocol presented here, the monophosphates and diphosphates of farnesol, geranylgeraniol and phytol are isolated from plant material with an isopropanol-containing buffer and quantified by LC-MS using citronellyl-P and citronellyl-PP as internal standards. With a low limit of detection for phytyl-P, geranylgeranyl-P, phytyl-PP, and geranylgeranyl-PP, isoprenyl-phosphates can be accurately measured in Arabidopsis leaves or seeds starting with only 20mg of fresh weight.