Knowing the structures and concentrations of lipids is crucial to identify biomarkers for diseases. Mass spectrometry (MS) has emerged as an important tool for lipid analysis due to its sensitivity and specificity. However, accurate relative quantification of lipids in biological samples has not been achieved due to the unavailability of commercial stable isotope labeled internal standards for each lipid in a complex sample to remove matrix effects on ionization efficiencies, not to mention quantitation of lipid isomers. To address these critical gaps, I have developed a novel two-step aziridine-based isobaric tag labeling strategy.

N-H aziridination generates an aziridine ring at the C=C bond position, which can be fragmented through ring opening upon collision-induced dissociation (CID) to generate two diagnostic ions, indicating the locations of C=C bonds. The introduction of trivalent “N” to lipids allows us to further functionalize lipid aziridines for accurate quantification via the amidation of aziridines with N-hydroxysuccinimide ester-based isobaric tags. Each of the two isobaric tags labels the lipids from disease and control groups at the aziridines, respectively. Same lipid shares identical m/z after labeling, followed by releasing charged mass reporters corresponding to lipids from different groups upon higher energy C-trap dissociation (HCD). The relative quantities of lipids from two samples are known by calculating the relative intensities of mass reporter ions. With the addition of lithium acetate, lithiated tagged lipids can generate sn-specific diagnostic ions for lipid sn-isomer identification upon CID. With further HCD, reporter ions from sn-diagnostic ions can be released for isomer quantification.

The strategy has been successfully demonstrated with unsaturated lipids from different categories, such as cholesterol esters (CE), fatty acids, ethyl oleate, and phospholipids. 17 CE lipid isomers were identified and quantified simultaneously from Alzheimer's disease (AD) mouse serum. Among them, 6 CE isomers showed significant changes in concentrations in AD serum.

Lipid Mass Tag Development for Isomer Analysis Via Aziridination.

Dallas Freitas

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