A New Approach to Detect and Semi-quantify All Molecular Species and Classes of Anionic Phospholipids Simultaneously in Plant Samples

Membranes are very complex and dynamic structures that are essential for plant cellular functions and whose lipidic composition can be influenced by numerous factors. Anionic phospholipids, which include phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphoinositides are key components of these membranes as they are involved in plant cell signaling and as even slight modifications in their quantities may largely impact the cell metabolism. However, the presence of these compounds in low amounts, as well as their poor stability during analysis by mass spectrometry, make their study very complicated. In addition, the precise quantification of all anionic phospholipid species is not possible by lipid separation using thin-layer chromatography followed by the analysis of their fatty acyl chains by gas chromatography. Here, we describe a straightforward strategy for the extraction and semi-quantification of all anionic phospholipid species from plant samples. Our method is based on the derivatization of the anionic phospholipids, and more especially on their methylation using trimethylsilyldiazomethane, followed by analysis by high-performance liquid chromatography coupled with a triple quadrupole mass spectrometer. This approach allows largely improving the sensitivity of the analysis of anionic phospholipids from plant samples, which will help to gain deeper insights into the functions and dynamics of these key parts of plant cellular signaling. Key features • Extraction, derivatization, and semi-quantification of anionic phospholipids (PA, PS, PI, PIPs) from plant samples. • The methylation of anionic phospholipids using trimethylsilyldiazomethane largely improves their stability and therefore the sensitivity of the analysis. • HPLC-QQQ-MS/MS analysis allows the semi-quantification of all methylated anionic phospholipids with high sensitivity. • Allows to investigate the diversity and dynamics of anionics phospholipids in a wide range of plant samples (leaves, cultivated cells, membranes, …).