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Shotgun lipidomics provides sensitive and fast lipid identification without the need for chromatographic separation. Challenges faced by shotgun analysis of glycerophospholipids (GPs) include the lack of signal uniformity across GP classes and the inability to determine the carbon-carbon double bond (C=C) location within the fatty acyl chains of an unsaturated species. Two distinct derivatization strategies were employed to both enhance the ionization of GPs, via trimethylation enhancement using (13)C-diazomethane ((13)C-TrEnDi), as well as determine location of double bonds within fatty acyl chains, employing an in-solution photochemical reaction with acetone (via the Paternò–Büchi reaction). The modified GPs were then subjected to positive ion mode ionization via electrospray ionization, producing uniform ionization efficiencies for different classes of GP species. The GPs were charge inverted via gas-phase ion/ion reactions and sequentially fragmented using ion trap collision-induced dissociation (CID). The CID of the species led to fragmentation producing diagnostic ions indicative of C=C bond location. The approach enabled enhanced ionization and the identification of phosphatidylcholine and phosphatidylethanolamine species at the C=C level in a bovine lipid extract.

Triacylglycerol (TG) is a class of lipids that is responsible for energy storage and cell metabolism in biological systems; it is found in relatively high abundances in biological fluids such as human plasma. Due to structural complexity, analyzing TGs using shotgun lipidomic approaches is challenging because of the presence of multiple fatty acyl compositional isomers. In this work, reversed-phase liquid chromatography (RPLC) was used for separation of TG species due to the capability of separating lipids based on fatty acyl chain lengths and degrees of unsaturation. RPLC alone does not provide structurally informative information for the location of carbon-carbon double-bonds (C[double bond, length as m-dash]Cs) without using synthesized standards that correspond to each species analyzed. The Paternò-Büchi (PB) reaction was employed online to confidently characterize the location of C[double bond, length as m-dash]Cs within lipid species via photo-initiated modification of the alkene group with acetone, which was later subjected to electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) to form signature fragmentation peaks. This online RPLC-PB-MS/MS system was able to distinguish fatty acyl level and C[double bond, length as m-dash]C level isomeric species. The systems allowed for the identification of 46 TG molecular species in human plasma with confident C[double bond, length as m-dash]C location assignment in fatty acyls at a limit of identification of 50 nM.

Shotgun lipid analysis based on electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is increasingly used in lipidomic studies. One challenge for the shotgun approach is the discrimination of lipid isomers and isobars. Gas-phase charge inversion via ion/ion reactions has been used as an effective method to identify multiple isomeric/isobaric components in a single MS peak by exploiting the distinctive functionality of different lipid classes. In doing so, fatty acyl chain information can be obtained without recourse to condensed-phase separations or derivatization. This method alone, however, cannot provide carbon-carbon double bond (C=C) location information from fatty acyl chains. Herein, we provide an enhanced method pairing photochemical derivatization of C=C via the Paternò-Büchi reaction with charge inversion ion/ion tandem mass spectrometry. This method was able to provide gas-phase separation of phosphatidylcholines and phosphatidylethanolamines, the fatty acyl compositions, and the C=C location within each fatty acyl chain. We have successfully applied this method to bovine liver lipid extracts and identified 40 molecular species of glycerophospholipids with detailed structural information including head group, fatty acyl composition, and C=C location. Graphical Abstract ᅟ.

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. Paternὸ-Buchi (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 coexisting 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.