Isomer Selective Comprehensive Lipidomics Analysis of Phosphoinositides in Biological Samples by Liquid Chromatography with Data Independent Acquisition Tandem Mass Spectrometry

Phosphoinositides (PIPx) play central roles in membrane dynamics and signal transduction of key functions like cellular growth, proliferation, differentiation, migration, and adhesion. They are highly regulated through a network of distinct phosphatidylinositol phosphates consisting of seven groups and three regioisomers in two groups (PIP and PIP2), which arise from phosphorylation at 3', 4', and 5' positions of the inositol ring. Numerous studies have revealed the importance of both fatty acyl chains, degree of phosphorylation, and phosphorylation positions under physiological and pathological states. However, a comprehensive analytical method that allows differentiation of all regioisomeric forms with different acyl side chains and degrees of phosphorylation is still lacking. Here, we present an integrated comprehensive workflow of PIPx analysis utilizing a chiral polysaccharide stationary phase coupled with electrospray ionization high-resolution mass spectrometry with a data independent acquisition technique using the SWATH technology. Correspondingly, a targeted data mining strategy in the untargeted comprehensively acquired MS and MS/MS data was developed. This powerful highly selective method gives a full picture of PIPx profiles in biological samples. Herein, we present for the first time the full PIPx profiles of NIST SRM1950 plasma, Pichia pastoris lipid extract, and HeLa cell extract, including profile changes upon treatment with potential PI3K inhibitor wortmannin. We also illustrate using this inhibitor that measurements of the PIPx profile averaged over the distinct regioisomers by analytical procedures, which cannot differentiate between the individual PIPx isomers, can easily lead to biased conclusions.

Automated summary

Phosphoinositides (PIPx) are crucial in cellular functions like growth, proliferation, differentiation, and are regulated by fatty acyl chains, degree of phosphorylation, and phosphorylation positions. However, there is a lack of comprehensive analytical method for distinguishing all regioisomeric forms with different acyl side chains and degrees of phosphorylation. This study presents a powerful method for analyzing PIPx profiles in biological samples using a chiral polysaccharide stationary phase and high-resolution mass spectrometry.