From Metabolite Profiling To Biomolecule Quantification: Modern Mass Spectrometry Techniques Applied To Biological Research

Abstract

Mass spectrometry (MS) is a versatile and powerful analytical technique that has revolutionized our ability to analyze complex biological systems. In this dissertation, we explore novel applications of various MS methodologies, including paper spray ionization (PSI), sandpaper spray ionization (SSI), matrix-assisted laser desorption/ionization (MALDI), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These techniques were applied to profile, detect, and quantify a wide range of metabolites and biomolecules across different biological contexts. At York University, we employed sandpaper spray ionization mass spectrometry for the rapid and comprehensive analysis of maple leaves infected with distinct fungal species. This approach demonstrated the capability of this high-throughput and solvent-efficient screening technique to differentiate fungal infections by analyzing directly from the leaf surface without requiring sample preparation. The study highlighted SSI-MS as a powerful tool for in situ metabolomic analysis of plant-pathogen interactions, enabling the detection of key metabolic changes associated with different fungal infections. Additionally, we developed a MALDI-high resolution MS method for the absolute quantification of the phytoalexins camalexin and scopoletin in Arabidopsis thaliana, providing novel insights into the plant’s biochemical responses to environmental stressors. Furthermore, a semi-quantitative version of this method was employed to perform relative quantification of a broader set of phytoalexins in wild-type and mutant Arabidopsis thaliana plants lacking key transcription factors involved in their production. In collaboration with Nucro-Technics Laboratories, we developed an ultra-sensitive immunoprecipitation-LC-MS/MS method for the quantification of low-abundance proteins in rat serum. This approach significantly enhances detection precision and sensitivity, allowing for the accurate quantification of trace-level proteins (≥ 1 ng/mL) in plasma while requiring small sample volumes (≤ 50 μL). This method meets the increasing demand for highly sensitive and reliable biomolecular assays in clinical and pharmaceutical research. These applications underscore the adaptability and efficacy of modern mass spectrometry in tackling diverse biological questions, ranging from plant-pathogen interactions to clinical biomolecule analysis. The methodologies developed and optimized in this dissertation provide valuable tools for MS-based metabolite profiling, biomolecule quantification, and the exploration of complex biological matrices. This research lays the foundation for future advancements in high-throughput screening, precision medicine, and plant metabolic studies.