Background

Anionic phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol (PI) species, as well as the phosphorylated form of the latter, namely monophosphate phosphoinositide (PIP) and bisphosphate phosphoinositide (PIP₂) molecules in plants. These compounds form a structurally diversified class of membrane lipids since a high variety of fatty acyl chains can be found esterified in these different negatively charged phospholipids, making their analysis very challenging. However, anionic phospholipids display key functions in plant cells as they are components of the plasma membrane and are involved in a variety of cellular processes. Notably, anionic phospholipids are essential components of plant membrane signatures, allowing their recognition by specific enzymes; they can also modulate their physicochemical properties (such as charge and curvature) in response to environmental features. These lipids also play important roles in signal transmission as their accumulation is very dynamic and regulated by many enzymes; they can be quickly degraded or metabolized for the production of secondary messengers [1].

While mass spectrometry, through a direct infusion approach or in combination with liquid chromatography, is generally the method of choice for lipidomic analysis, the low abundance of anionic phospholipids as well as their poor stability and ionization efficiency during analysis increase the difficulty of their profiling and quantification, notably from plant samples. Indeed, sensitivity problems as well as low peak resolution are highlighted. For this reason, a multi-step approach is generally used for the analysis of anionic phospholipids by combining the separation of their different classes by preparative thin-layer chromatography and the analysis of their fatty acyl chains by gas chromatography. While this method allows us to understand the diversity in the fatty acyl chains of each class of anionic phospholipids, it does not give a comprehensive view of the diversity of anionic phospholipids in plant samples.

Based on the fact that methylation improves anionic lipid ionization efficiency, we developed a straightforward method for the extraction and semi-quantification of all classes of anionic phospholipids (Table S1) from plant samples by HPLC–MS/MS following their methylation using trimethylsilyldiazomethane [2]. Our results highlight not only the successful application of this method to a large set of plant samples (e.g., leaves, roots, purified membranes, and cultivated cells) but also the great improvement in the sensitivity of anionic phospholipids analyses in comparison with the same lipids analyzed without being methylated. Notably, methylation improved the wide peak shape and therefore enhanced the precision of the analysis. Our method therefore paves the way for the development of straightforward methods allowing the profiling and quantification of anionic phospholipids with high precision and sensitivity, not only from plant samples but also from other biological materials.