<i>sn</i>-Position-Resolved Quantification\nof Aminophospholipids by Isotopic <i>N</i>,<i>N</i>‑Dimethyl Leucine Labeling and High-Resolution Ion Mobility\nMass Spectrometry

Aminophospholipids (APLs), composed of phosphatidylethanolamines\n(PEs) and phosphatidylserines (PSs), are vital components of mammalian\ncell membranes and lipoproteins, participating in both homeostasis\nand cellular signaling. Their structural changes, including the permutation\nof fatty acid connectivity (<i>sn</i>-positions), due to\ndysfunctional metabolic processes have been linked to many diseases.\nHowever, the accurate quantification of APLs with unambiguous fatty\nacyl assignment through routine label-free LC–MS/MS lipidomic\nanalysis remains a major challenge. In this study, we explore the\nfunctionalization of the free primary amine groups of APLs using amine-reactive\nisotopic <i>N</i>,<i>N</i>-dimethyl leucine (iDiLeu)\nand employ high-resolution ion mobility MS (IM-MS) to develop a novel\nmethod for sensitive discernment and accurate quantification of APL <i>sn</i>-isomers. With high-resolution demultiplexing (HRdm) providing\nIM resolving power >200, labeled <i>sn</i>-isomeric pairs\nof APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline\nseparation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu\nlabeling enables the construction of an internal 4-point calibration\ncurve and therefore absolute quantification of APL <i>sn</i>-isomers in a single run. This strategy enabled precise annotation\nand quantification of 239 APLs including 60 pairs of <i>sn</i>-isomers in the mouse cortex. Additionally, we were able to find\nratio changes in multiple APL <i>sn</i>-isomer pairs between\nwild type and APP/PS1 Alzheimer’s disease (AD) model mice at\ndifferent ages, indicating their strong correlation to AD progression.\nThis strategy could provide universal utility in unraveling the alteration\nof APL <i>sn</i>-isomers, which have long been considered\nas the “dark matter” of traditional lipidomic analyses,\nleading to more precise elucidation of molecular mechanisms of various\ndiseases.