PhD Seminar - Ritchie Ly, September 23, 3:30
Title: Strategies for Expanding Lipid Coverage and Accelerating Biomarker DiscoveryUsing Multisegment Injection-Nonaqueous-Capillary Electrophoresis-MassSpectrometry
Date: Friday, September 23, 2022
Zoom: For Zoom details please email firstname.lastname@example.org
Host: Philip Britz-McKibbin
Abstract: The importance of biomarkers cannot be understated as theyhave played a key role in revolutionizing public health and disease preventionon a population level. While there is a need to discover more specificbiomarkers, many technical challenges exist especially when characterizingunknown lipids of clinical significance from small volumes of blood. Thisthesis aims to develop new analytical strategies in clinical medicine fornontargeted lipid profiling (i.e., lipidomics) when using multisegment injection-nonaqueouscapillary electrophoresis-mass spectrometry (MSI-NACE-MS). Chapter II introducesa nontargeted approach to identify new biomarkers for galactosemia from asingle neonatal dried blood spot punch extract as an alternative toconventional enzyme assays prone to false positives. The use of modified sampleadapters allowed for reproducible analysis of volume-limited specimens (< 2µL), whereas multiplexed CE-MS separations with temporal signal patternrecognition facilitated the identification of biomarker candidates by comparingthe metabolic phenotype of different disease variants of galactosemia andhealthy neonatal controls within a single experimental run. However, analyseswere limited to polar/ionic metabolites when using aqueous-based separationsystems. Chapter III greatly expands metabolome coverage in CE-MSwhen using a compatible non-aqueous electrolyte system for global analysis ofionic lipids differing widely in their polarity, such as lysophosphatidicacids, phosphatidylinositols, phosphatidylethanolamines and nonesterified fattyacids. For the first time, a multi-tiered data workflow was introduced inMSI-NACE-MS using an ultra-high resolution Orbitrap mass analyzer undernegative ion mode for credentialing more than 270 lipid features from serumextracts based on their characteristic accurate mass and electrophoreticmobility. Of these, 128 anionic lipids were reliably measured (median CV ≈ 13%)in most serum extracts (> 75%) that were applied to stratify a cohort ofnonalcoholic steatohepatitis patients (n = 85) based on diseaseseverity. Chapter IV expands the analytical performance of MSI-NACE-MSby introducing an innovative two-step chemical derivatization strategy to affixa permanent positive charge on zwitter-ionic lipids, such asphosphatidylcholines, sphingomyelinsand plasmalogens. Quantitative chemical labelingallowed for improved resolution and detectability of permethylated lipidcations, which was evaluated on a standard serum reference material (NIST 1950)to compare overall lipid coverage and reported concentrations from aninternational lipidomics ring trial. This chemical labeling strategy may alsoprove useful in other lipidomic platforms (LC-MS/MS, DI-MS/MS) since it iscost-effective and less hazardous than diazomethane. Lastly, Chapter Vapplied MSI-NACE-MS with lipid permethylation to discover novel biomarkersassociated with omega-3 index in two independent fish oil supplementationclinical trials involving the ingestion of omega-3 fatty acids eicosapentaenoicacid and/or docosahexaenoic acid. For the first time, we identified a panel ofomega-3 containing phosphatidylcholines in serum extracts that displayedselective treatment responses in participants following fish oilsupplementation as compared to placebo. This work revealed that specificcirculating phospholipids may pave the way for rapid assessment of omega-3index and diet quality in large-scale epidemiological studies relative toclassic erythrocyte membrane lipid measurements. Overall, this thesis introducesMSI-NACE-MS as a hitherto unrecognized analytical platform for lipidomics thatis complementary to chromatographic methods with improved sample throughput andaccelerated data workflows for biomarker discovery in clinical medicine.