Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.
Keywords: Animal models; Lipid signaling; Lipid storage; Lipidomics; Phosphatidylethanolamines; Pre-analytical methods.