Aziridination-Assisted Mass Spectrometry of Nonpolar Sterol Lipids with Isomeric Resolution

Characterization of nonpolar lipids is crucial due totheir essentialbiological functions and ability to exist in various isomeric forms.In this study, we introduce the N-H aziridination method totarget carbon-carbon double bonds (C C bonds) in nonpolarsterol lipids. The resulting fragments are readily dissociated uponcollision-induced dissociation, generating specific fragment ionsfor C C bond position determination and fingerprint fragmentsfor backbone characterization. This method significantly enhanceslipidionization efficiency, thereby improving the sensitivity and accuracyof nonpolar lipid analysis. We demonstrated that aziridination ofsterols leads to distinctive fragmentation pathways for chain andring C C bonds, enabling the identification of sterol isomerssuch as desmosterol and 7-dehydrocholesterol. Furthermore, aziridinationcan assist in identifying the sterol backbone by providing fingerprinttandem mass spectra. We also demonstrated the quantitative capacityof this approach with a limit of detection of 10 nM in the solventmixture of methanol and water. To test the feasibility of this methodin complex biological samples, we used mouse prostate cancerous tissuesand found significant differences in nonpolar lipid profiles betweenhealthy and cancerous samples. The high efficiency and specificityof aziridination-assisted mass spectrometric analysis, as well asits quantitative analysis ability, make it highly suitable for broadapplications in nonpolar lipid research.