Lipidome-wide characterization of phosphatidylinositols and phosphatidylglycerols on C=C location level

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) are two essential classes of glycerophospholipids (GPs), playing versatile roles such as signalling messengers and lipid-protein interaction ligands in cell. Although a majority of PG and PI molecular species contain unsaturated fatty acyl chain(s), conventional tandem mass spectrometry (MS/MS) methods cannot discern isomers different in carbon-carbon double bond (C=C) locations. In this work, we paired phosphate methylation with acetone Paternò–Büchi (PB) reaction, aiming to provide a solution for sensitive and structurally informative analysis of these two important classes of GPs down to the location of C=C. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was established. Offline methylated PG or PI mixtures were subjected to hydrophilic interaction chromatographic separation, online acetone PB reaction, and MS/MS via collision-induced dissociation (CID) for C=C location determination in positive ion mode. This method was sensitive, offering limit of identification at 5 nM for both PG and PI standards down to C=C locations. On molecular species level, 49 PI and 31 PG were identified from bovine liver, while 61 PIs were identified from human plasma. This workflow also enabled ratiometric comparisons of C=C location isomers (C18:1 Δ9 vs. Δ11) of a series of PIs from type 2 diabetes (T2D) plasma to that of normal plasma samples. PI 16:0_18:1 and PI 18:0_18:1 were found to exhibit significant changes in C=C isomeric ratios between T2D and normal plasma samples. The above results demonstrate that the developed LC-PB-MS/MS workflow is applicable to different classes of lipids and compatible with other established lipid derivatization methods to achieve comprehensive lipid analysis.

• This study applies phosphate methylation to enhance identification of phosphatidylglycerol (PG) and phosphatidylinositol (PI) at the C=C location level using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow coupled with online Paternò-Büchi (PB) reaction.
• The developed workflow reveals significant changes in C=C location isomers of PI 16:0_18:1 and PI 18:0_18:1 in plasma samples between type 2 diabetes patients and normal controls, demonstrating the applicability of HILIC-PB-MS/MS to various classes of lipids and compatibility with other derivatization methods.