Strategies for Expanding Lipid Coverage and Accelerating Biomarker Discovery Using Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry

Abstract

The importance of biomarkers cannot be understated as they have played a key role in revolutionizing public health and disease prevention on a population level. While there is a need to discover more specific and clinically relevant biomarkers, many technical challenges exist especially in the context of the rapidly growing field of lipidomics. This thesis aims to develop new analytical strategies for nontargeted lipid profiling when using multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). Chapter II greatly expands metabolome coverage in CE-MS when using a compatible non-aqueous electrolyte system for global analysis of ionic lipids differing widely in their polarity, such as lysophosphatidic acids, phosphatidylinositols, phosphatidylethanolamines and free fatty acids. For the first time, a multi-tiered data workflow was introduced in MSI-NACE-MS using an ultra-high resolution Orbitrap mass analyzer under negative ion mode for credentialing more than 270 lipid features from serum extracts based on their characteristic accurate mass and electrophoretic mobility. Of these, 128 anionic lipids were reliably measured (median CV ≈ 13%) in most serum extracts (> 75%) that were applied to stratify a cohort of Japanese non-alcoholic steatohepatitis patients (n = 85) based on their disease severity not feasible by conventional biomarkers of liver fibrosis. Chapter III expands the analytical performance of MSI-NACE-MS by introducing an innovative two-step chemical derivatization protocol as a charge-switching strategy to resolve zwitter-ionic phospholipids that otherwise co-migrate with the electroosmostic flow. Reaction conditions were optimized to achieve a quantitative yield for methylated phosphatidylcholines, which greatly improved resolution and detectability when using MSI-NACE-MS under positive ion mode. Overall, this approach expanded lipidome coverage, supported phospholipid structural identification based on their characteristic electrophoretic mobility while also demonstrating the potential for reliable quantitative determination in a human serum reference material (NIST SRM-1950). Importantly, this universal methylation strategy may prove useful in other lipidomic platforms (LC-MS/MS, DI-MS/MS) as it is less hazardous than diazomethane. Lastly, Chapter IV involved the application of MSI-NACE-MS technology to discover surrogate biomarkers of omega-3 index (O3I) in two independent placebo-controlled clinical trials involving the ingestion of high-dose omega-3 fatty acids, including fish oil, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). For the first time, we identified a panel of omega-3 containing phosphatidylcholines in serum and plasma ether extracts that exhibited treatment responses in participants that were also positively correlated with independent O3I measurements. This work revealed that specific circulating phospholipids may allow for rapid assessment of omega-3 index without the need for complicated erythrocyte membrane hydrolyzed fatty acid analysis as required for risk assessment of cardiovascular health and sudden cardiac death. Overall, this thesis introduced MSI-NACE-MS as a hitherto unrecognized analytical platform for lipidomics that is complementary to chromatographic methods with improved sample throughput and accelerated data workflows for biomarker discovery in clinical medicine.