PE, or not PE, that is the question: The case of overlooked lyso-N-acylphosphatidylethanolamines

Rationale Lyso derivatives of N-acyl-1,2-diacylglycero-3-phosphoethanolamines (L-NAPEs) are a lipid class mostly expressed in vegetables during stress and tissue damage that is involved in the synthesis of the lipid mediator N-acylethanolamines. L-NAPEs can be challenging to distinguish from isomeric phosphatidylethanolamines (PEs), especially in extracted complex samples where they could be confused with abundant PEs. Methods In this study, hydrophilic interaction liquid chromatography with electrospray ionization hyphenated with (tandem) mass spectrometry (MS) was proposed to distinguish L-NAPEs and PEs as deprotonated molecules, [M − H]─, using both high-resolution/accuracy Fourier transform MS and low-resolution linear ion trap (LIT) mass analyzers. MS3 experiments of [M − H − KE]─ as precursor ions (KE, ketene loss) using the LIT instrument allowed us to distinguish between isomeric L-NAPE and PE species. Results Regiochemical rules were proposed working on enzymatically synthesized L-NAPEs. A few key differences in MS/MS spectra, including abnormal intensity of acyl chain losses as fatty acids, the presence of N-acylphosphoethanolamine ions, and diagnostic ions of the polar head, were disclosed. Additionally, MS3 spectra of [M − H − KE]─ as precursor ions allowed us to confirm the identification of L-NAPE species. The proposed rules were applied to samples extracted from tomato by-products including stems and leaves. Conclusions Overall, our methodology is demonstrated as a robust approach to recognizing L-NAPEs in complex samples. L-NAPEs 18:2-N-18:2, 18:2-N-18:3, 18:3-N-18:2, and 18:2-N-18:1 were the prevailing compounds in the analyzed tomato samples, accounting for more than 90%. In summary, a reliable method for identifying L-NAPEs in complex samples is described. The proposed method could prevent overlooking L-NAPEs and overestimating isomeric PE species in future lipid analyses.