Uncovering Structural Diversity of Unsaturated Fatty Acyls in Cholesteryl Esters via Photochemical Reaction and Tandem Mass Spectrometry

Abstract

Mass spectrometry analysis of cholesteryl esters (CEs) faces several challenges with one of them being the determination of the carbon-carbon double bond (C=C) locations within unsaturated fatty acyl chains. Paternò-Büchi (PB) reaction, a photochemical reaction based on the addition of acetone to C=C, is capable of C=C location determination when coupled with tandem mass spectrometry (MS/MS). In this study, the PB reaction conditions were tailored for CEs and subsequent nanoelectrospray ionization (nanoESI). A solvent system containing acetone/methanol/dichloromethane/water (40/30/20/10, volume ratios) and 100 μM LiOH was determined to be optimal, resulting in reasonable PB reaction yield (~30%) and good ionization efficiency (forming lithium adduct of CEs). Collision-induced dissociation (CID) of the PB reaction products produced characteristic fragment ions of CE together with those modified by the PB reactions, such as lithiated fatty acyl ([FA+Li]+) and its PB product ([FA-PB+Li]+). MS3 CID of [FA-PB +Li]+ led to abundant C=C diagnostic ion formation, which was used for C=C location determination and isomer quantitation. A PB-MS3 CID approach was developed and applied for CE analysis from human plasma. A series of unsaturated CEs was identified with specific C=C locations within fatty acyl chains. Absolute quantitation for each CE species was achieved including co-existing C=C location isomers, such as Δ9 and Δ11 isomers of CE 18:1 and ω-6 and ω-3 isomers of CE 18:3. These results show that PB-MS/MS is useful in uncovering structural diversity of CEs due to unsaturation in fatty acyls, which is often undetected from current lipid analysis approach.

Recent advances in lipid separations and structural elucidation using mass spectrometry combined with ion mobility spectrometry, ion-molecule reactions and fragmentation approaches

“…These cleavages form a new lipid and an aldehyde/ketone diagnostic pair with a mass difference of 26 Da, which can be used to identify the C=C bond locations. P-B reactions coupled with CID have been successfully utilized for the identification and quantitation of unsaturated lipids in lipidomics studies and have even illustrated the importance of C=C positions in patients with breast cancer [59–63]. …”

Collision-induced dissociation of doubly-charged barium-cationized lipids generated from liquid samples by atmospheric pressure matrix-assisted laser desorption/ionization provides structurally diagnostic product ions

“…Relative and absolute quantitation of constituent isomers has been reported based on the intensity of the diagnostic product ions [30]. Recently, this characteristic Δm/z of 26 has been applied to double bond determination in cholesteryl ester lithium adducts by MS 3 analysis of the PB-reaction products [31]. The possibility of including the carbonyl reagent in the LC mobile phase makes PB-MS a good option for online lipidomic workflows.…”

Double bond localization in unsaturated rhamnolipid precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids by liquid chromatography–mass spectrometry applying online Paternò–Büchi reaction

“…Since 2014, a number of related approaches was presented and successfully applied to a variety of biological samples [53][54][55][56][57][58]. Moreover, modifications of the initially introduced setup were reported, tackling particular drawbacks of this approach.…”