Developing A Mass Spectrometry Workflow For High-sensitivity And High-coverage Analysis Of Lipidome Molecular Structures

Lipids represent crucial metabolites within organisms,intimately associated with diverse biological processes,including cellular membrane biogenesis,signal transduction,and energy metabolism.In biological systems,lipid molecules exhibit a remarkable structural diversity,characterized by the presence of multiple isomers,encompassing fatty acyl chain isomers,sn-position isomers,and carbon-carbon double bond(C=C)location isomers.Nonetheless,elucidating the structures of these isomers poses formidable challenges.Currently,tandem mass spectrometry(MS/MS)via collisioninduced dissociation(CID)enables to identify the fatty acyl chain composition,yet falls short in locating the position of C=C within unsaturated acyl chains.This limitation impedes us to understand lipid metabolism and its associated functions at the molecular species level.To overcome the intricate task of detailed structure analysis of lipid,this doctoral dissertation proposes a novel solution and establishes a qualitative and quantitative analytical methodologies,tailored for glycerolipids and phospholipids at the detailed structure level.Specifically,this study delves into five principal dimensions:(1)The implementation of phosphate methylation reaction addresses the issue of low ionization efficiency encountered with phosphatidylglycerol(PG)and phosphatidylinositol(PI)in positive ion mode.Furthermore,the online acetone Paternò-Büchi(PB)reaction enables the detailed structural analysis of PG and PI at the C=C location level.(2)The employment of charge-tagged PB derivatization reagent(2-acetylpyridine)enhances the sensitivity of qualitative and quantitative analysis of glycerides at the C=C location level.The incorporation of this methodology into supercritical fluid chromatography(SFC)and ion mobility mass spectrometry enables accurate structural analysis of glycerides in plasma samples on a large scale.(3)The integration of hydrophilic interaction chromatography(HILIC)and trapped ion mobility spectrometry(TIMS),offering orthogonal separation and complementary advantages,coupled with isomer-resolved tandem mass spectrometry,establishes a highly efficient and comprehensive analytical system for profiling phospholipidomes.This system possesses several remarkable features,including rapid analysis,exceptional sensitivity,and wide coverage.The performance of this analysis system has been demonstrated through successful applications to bovine liver and RAW 264.7 cell samples,leading to the identification of more than 1500 unique molecular structures of phospholipids.Furthermore,the system enables accurate monitoring of compositional changes among diverse phospholipid isomers in human bladder cancer tissue.With its capability for comprehensive lipidomic analysis,this system serves as a valuable tool for investigating lipid metabolism,conducting phenotype analysis,and facilitating various biomedical applications.(4)The application of charge-tagged Paternò-Büchi(PB)derivatization in combination with desaturase inhibition enables the discovery of novel unsaturated fatty acid sites and potential metabolic pathways in RAW 264.7 macrophages.(5)The incorporation of isomer-resolved tandem mass spectrometry into a Capillary reversed-phase chromatography-TIMS-MS/MS system achieves significant sensitivity enhancement at the sub-nanomolar per liter level.