<i>sn</i>-Position-Resolved Quantification of Aminophospholipids by Isotopic <i>N</i>,<i>N</i>-Dimethyl Leucine Labeling and High-Resolution Ion Mobility Mass Spectrometry

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity (<i>sn</i>-positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic <i>N</i>,<i>N</i>-dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL <i>sn</i>-isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled <i>sn</i>-isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL <i>sn</i>-isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of <i>sn</i>-isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL <i>sn</i>-isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL <i>sn</i>-isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.