Development of Mass Spectrometric Methods for Analysis of Sphingolipids and Oligonucleotides

Abstract

Mass Spectrometry (MS) is a powerful method for analysis of biomolecules due to its selective and sensitive analytical benchmarks, providing information about their structures and abundance, further enabling their functions to be studied. This thesis focuses on the analysis of sphingolipids and oligonucleotide therapeutics due to knowledge gaps in their MS analysis. Sphingolipids (SPs) are pivotal membrane lipids with very diverse structures, setting the stage for challenging analysis. To enhance analytical performance, lithium was incorporated into the MS workflow, consolidating adducts and simplifying the mass spectrum and proving informative fragmentation patterns. An extraction protocol was developed that integrated a base hydrolysis step for SP enrichment using lithium hydroxide, which effectively hydrolyzed esterified lipids with the added benefit of lithium adduct consolidation. A high throughput screening method was developed with lithium adduct consolidation, resulting in detection enhancement of low abundant SPs. A multidimensional analytical platform was developed to provide higher structural quality of SPs, utilizing off-line liquid chromatograph (LC), ion mobility and high-resolution tandem MS. The off-line LC provided separation and allowed lithium adduction for further analysis while ion mobility and elevated energy tandem MS are used to structurally characterize and resolve SP isomers. Data processing, analysis and visualizations techniques were also developed, tailored to the specific needs of the workflow. A MS imaging method for spatial localization of SPs was developed with lithium adduction and on tissue hydrolysis to enhance SP analysis of intact tissue. OGN (oligonucleotide) therapeutics are becoming increasingly more popular for complex diseases. Despite this, rigorous analytical techniques to monitor biomanufacturing processes and the final formulation product are lacking. A high-throughput screening method was developed to verify the molecular weight and to scan for non-isomeric impurities while minimizing alkali salt contamination that notoriously adduct to OGNs during ionization. LC methods were developed for both analytical and preparative separation while tandem MS was used to confirm their sequence. For isomeric impurities, ion mobility was utilized to interrogate and compare the extremely complicated diastereomeric composition of various OGN drug products.