File(s) under embargo
until file(s) become available
Biomedical Testing by Direct Sampling Ionization with Miniature Mass Spectrometry Systems
Biomedical testing by mass spectrometry (MS) in clinical laboratories is fundamental to providing clinicians with accurate information to confirm their initial diagnoses. However, laboratory-based testing requires careful handling, transport, and complex sample analysis to achieve results with appropriate sensitivities. Patient results are reviewed during the next schedule appointment, hindering the initiation of treatment and adversely affecting patient health outcomes. The introduction of miniature MS systems and its related studies have introduced a basic outline for the implementation of a point-of-care (POC) biomedical testing tool. Current miniature MS systems have been applied to analyzing and monitoring therapeutic drugs and drugs of abuse, using simple sampling procedures. As biomedical testing begins to shift towards analyzing biomolecules, this dissertation seeks to further the applicability of direct sampling ionization with miniature MS systems. Biomolecules with current and emerging diagnostic significance such as proteins, metabolites, and lipids were analyzed using a miniature MS system, integrating both conventional and novel sampling methods.
The first study introduces a protein biomarker analytical workflow using a miniature MS system tied to an immunoaffinity enrichment protocol. A dual linear ion trap miniature MS system was optimized to quantify peptides in solution across a wide mass range, performing high-efficiency tandem MS at a relatively high sensitivity. Amino acid sequences of the digested peptides were identified using several types of collision-induced dissociation (CID). Quantitation of peptides was performed within a solution matrix of similarly digested peptides through the incorporation of internal standards (IS) and product ion monitoring. Finally, the entire workflow was tested by quantifying the targeted Met peptide sequence from cell line with a low Met protein expression level.
The second study establishes a workflow for lipid profiling of biofluids using a novel direct sampling ionization method with our miniature MS system. Downstream from proteins, lipids represent a class of metabolic biomolecules that directly reflect the biological state. Metabolic diseases cause a distinct perturbation from the norm that is reflected in the lipid profile acquired from comprehensive extraction and analysis. Polymer-coating transfer enrichment was developed to improve the extraction efficiency of lipids from biofluids while eliminating the sample matrix in less than a minute. Photochemical reactions were combined with the novel direct sampling method for enhanced lipid structure elucidation. Preliminary investigations into the free fatty acid profile of healthy and Type-2 diabetes human patient plasma samples was performed, resulting in several distinct profiles for disease differentiation.
The final study builds a workflow to analyze exogenous metabolites, specifically mycotoxins produced by fungi, in feed and foodstuffs. Mycotoxins pose a significant concern to the world’s grain storages, emphasizing the need for constant monitoring to minimize mycotoxin exposure and ingestion. By combining slug-flow microextraction with a miniature MS system, four different mycotoxins were analyzed in different matrices. A surface analysis technique was also proposed, eliminating the need for initial sample preparation before analysis. Trace amounts of mycotoxins could be detected from the surface of a corn kernel without sample destruction. Thus, a universal workflow for continuous monitoring of mycotoxins in grain storages worldwide was outlined in this study.