Lipids play a pivotal role in biological processes and lipid analysis by mass spectrometry (MS) has significantly advanced lipidomic studies. While the structure specificity of lipid analysis proves to be critical for studying the biological functions of lipids, current mainstream methods for large-scale lipid analysis can only identify the lipid classes and fatty acyl chains, leaving the C=C location and sn-position unidentified. In this study, combining photochemistry and tandem MS we develop a simple but effective workflow to enable large-scale and near-complete lipid structure characterization with a powerful capability of identifying C=C location(s) and sn-position(s) simultaneously. Quantitation of lipid structure isomers at multiple levels of specificity is achieved and different subtypes of human breast cancer cells are successfully discriminated. Remarkably, human lung cancer tissues can only be distinguished from adjacent normal tissues using quantitative results of both lipid C=C location and sn-position isomers.

Large-scale profiling of phosphatidylcholines at the isomer level is achieved by incorporating gas-phase radical-directed fragmentation into an LC-MS/MS workflow.

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

The presence of carbon-carbon double bonds (C═Cs) in unsaturated phospholipids is closely related to lipid conformations and physiochemical activities. Previously, we have demonstrated that epoxidation reaction facilitated by low-temperature plasma (LTP) enabled the structural analysis of unsaturated fatty acids (FAs). Epoxidation of the C═C leads to the production of an epoxide, which can be easily cleaved via collision-induced dissociation (CID) to produce diagnostic ions indicative of the C═C bond locations in FAs. In this work, we further developed this method for analysis of phospholipids. Tandem mass spectrometry analysis with epoxidation reaction was performed in both positive and negative ion mode to analyze phosphatidylcholines (PCs), phosphatidic acids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The developed method was applied in a shotgun lipidomics approach to characterize phospholipids in a bovine liver extract.

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The [2 + 2] photocycloaddition of alkenes and carbonyls is of fundamental interest and practical importance, as this process is extensively involved in oxetane-ring constructions. Although individual carbonyl group or alkene moiety has been utilized as photoactive species for oxetane formations upon ultraviolet photoexcitation, direct excitation of the entire noncovalent complex involving alkene and carbonyl substrates to achieve [2 + 2] photocycloadditions is rarely addressed. Herein, complexes with noncovalent interactions between benzophenone and CC bonds in unsaturated lipids have been successfully characterized, and for the first time a [2 + 2] cycloaddition leading to the formation of oxetanes has been identified under visible-light irradiation. The mechanism of this reaction is distinctly different from the well-studied Paternò–Büchi reaction. The entire complexes characterized as dimeric proton-bonded alkene and carbonyl substrates can be excited under visible light, leading to electron transfer from the alkene moiety in fatty acyls to the carbonyl group within the complex. These results provide new insight into utilizing noncovalent complexes for the synthesis of oxetanes in which the excitation wavelength becomes independent of each individual substrate.

Mass spectrometry imaging (MSI) serves as a powerful tool for biological research, and laser desorption ionization (LDI) is used as a major sampling ionization method. Study of materials for LDI represents a major field in the MSI research, either for matrices in matrix-assisted LDI (MALDI) or sample substrates allowing matrix-free LDI. In this study, we developed a composite substrate using polydopamine (PDA) film to coat an antireflection (AR) surface for LDI-MSI. The AR material has been previously shown to confine UV energy within the micro-/nanostructures, leading to a highly localized temperature rise to facilitate analyte thermal desorption. PDA coating on the AR material further enhances the light-to-heat conversion and improves the contact between the substrate surface and the biological sample materials. With this substrate, desorption and ionization of lipids from raw human plasma samples and biological tissue sections have been achieved. Matrix-free LDI-MSI of around 30 lipid species in mouse brain sections was achieved with a significantly simplified MSI procedure at a spatial resolution of 50 μm. This method was applied to determine mouse fatty liver disease through monitoring the abundances and distributions of triacylglycerols and glycerophospholipids. Dramatic differences in the lipid profiles were subsequently identified between the liver tissues from the wild-type and obese mice.

The combination of direct sampling ionization and miniature mass spectrometer presents a promising technical pathway of point-of-care analysis in clinical applications. In this work, a miniature mass spectrometry system was used for analysis of tissue samples. Direct tissue sampling coupled with extraction spray ionization was used with a home-built miniature mass spectrometer, Mini 12. Lipid species in tissue samples were well profiled in rat brain, kidney, and liver in a couple of minutes. By incorporating a photochemical (Paternò–Büchi) reaction, fast identification of lipid CC location was realized. Relative quantitation of the lipid CC isomer was performed by calculating the intensity ratio CC diagnostic product ions, by which FA 18:1 (Δ9)/FA 18:1 (Δ11) was found to change significantly in mouse cancerous breast tissue samples. Accumulation of 2-hydroxylglutarate in human glioma samples, not in normal brains, can also be easily identified for rapid diagnosis.