Enabling High Structural Specificity to Lipidomics by Coupling Photochemical Derivatization with Tandem Mass Spectrometry

CONSPECTUS: Lipids have pivotal roles in many biological processes, including energy storage, signal transduction, and plasma membrane formation. A disruption of lipid homeostasis is found to be associated with a range of diseases, such as cardiovascular diseases, diabetes, and cancer. Fundamental lipid biology and disease diagnostics can benefit from monitoring lipid changes in cells, tissues, organs, or the whole biological system. Therefore, it is important to develop lipid analysis tools to achieve comprehensive lipid characterization and quantitation. Over the past two decades, mass spectrometry (MS) has become the method of choice for qualitative and quantitative analyses of lipids, owing to its high sensitivity, multiplexed analysis, and soft ionization features. With the rapid development and adoption of ultrahigh-resolution MS, isobaric lipids can now be routinely resolved. By contrast, the structural characterization and quantitation of isomeric lipids remain an analytical challenge. Although some lipid C = C location or sn- isomers can be resolved by chromatography, ion mobility, or selective ionization approaches, a detailed structural characterization on the lipidome-wide level needs to be achieved. Over the past six years, we have successfully combined the Paterno-Buchi (PB) reaction, which is a UV-promoted photocycloaddition reaction specific to the C = C, with tandem MS (MS/MS) to locate the C = C in lipids and quantify lipid C = C location isomers. The PB reactions have analytical advantages such as a simple experimental setup, rapid lipid C = C derivatization, and highly specific C = C cleavage during PB-MS/MS to produce abundant diagnostic ions. More importantly, without a need of isomer separation or a comparison to authentic standards, PB-MS/MS can be directly applied to identify and quantify a mixture of lipid C = C location isomers, often coexisting with molar ratios sensitive to the biological state of the system. The PB-MS/MS method is compatible with conventional shotgun lipidomics employing a nanoelectrospray ionization or a large-sale lipid structural analysis via liquid chromatography (LC) coupled to any mass spectrometer with tandem MS capability. The PB-MS/ MS method is highly versatile, as a variety of PB reagents can be tailored to a broad range of applications. Besides UV-promoted PB reactions, visible-light PB reactions have also been developed to offer more flexibility for a lipid analysis. By using selected PB reagents, the sn-positions of fatty acyls can be resolved together with C = C locations in phospholipids. This method has been used in lipidomic analyses of tissue, blood, and plasma from animal models and clinical samples, demonstrating the potential of using lipid C = C or sn-location isomer ratios for phenotyping and disease diagnostics. Lipid isomer-resolving MS imagings of tissues and singlecell lipid analysis have also been demonstrated by a proper implementation of PB-MS/MS.

Automated summary

Lipids play crucial roles in biological processes and the precise determination of the location and quantity of lipid chains is essential for understanding biological systems. Mass spectrometry has become a powerful tool for lipid analysis, but the structural characterization and quantitation of lipid isomers remain challenging.