Development of High-Resolution Ion Mobility and Tandem Mass Spectrometry Approaches for Advanced Metabolomic Characterization of Eukaryotic Pathogenic Systems

Clemson University, Clemson SC

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Abstract

Eukaryotic pathogens threaten public health in the US, accentuated by ease of dissemination, difficulty in diagnosis, and/or emergence of common drug resistance. Two concerning threats include Naegleria fowleri and Aspergillus fumigatus. N. fowleri, the “brain-eating amoeba”, is found in warm freshwater and can cause primary amebic meningoencephalitis (PAM). However, PAM progresses rapidly and diagnostic laboratory tests are not widely available, resulting in a >97% fatality rate. On the other hand, A. fumigatus is a respiratory fungus especially dangerous to those with weakened immune systems. Inadequate diagnostic procedures lead to empiric drug administration and the associated rise of resistance. In both cases, the metabolomic effects of infection and/or their treatment are still not well characterized due to the biological complexity of these systems; specifically, challenges in sensitivity/dynamic range, breadth of metabolite polarity, and isomeric heterogeneity complicate analyses and often prompt very narrowly focused studies (i.e., of a particular molecular subclass as opposed to the global metabolome). Nevertheless, elucidating the metabolomic determinants of these infections promises improved development of therapeutics. Advances in ionization sources, mass analyzers, and associated techniques like chromatographic separations and ion mobility (IM) spectrometry have led to acquisition of biological datasets that are richer in both qualitative and quantitative data. Our long-term goal is to develop advanced technologies for determining the global metabolomic underpinning of eukaryotic pathogenic infections and associated changes resulting from therapeutic drug treatments. Our objective here is to integrate multidimensional chromatographic separations, high-resolution ion mobility (HRIM), and mass spectrometry imaging (MSI) for metabolomic studies of amoebic (i.e., Naegleria fowleri) and respiratory fungal pathogens (i.e., Aspergillus). Our central hypothesis is that these hyphenated measurements will collectively expand our breadth/depth of analysis by advancing molecular identification, quantification, and spatial localization, therefore enabling critical biological insights that will ultimately lead to improvements in diagnosis and treatment. The objective of this proposal will involve pursuing the following two specific aims: 1. Identify metabolomic differences in pathogen-infected and drug-treated models relative to healthy controls using two-dimensional chromatography coupled to high-resolution ion mobility-mass spectrometry (HRIMMS). Our hypothesis is that this depth will provide more comprehensive identification of metabolomic signatures representative of infection and/or treatment. 2. Determine spatial localization of metabolomic biomarkers and drugs in biological tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging with high resolution tandem mass spectrometry (HRMS/MS) and targeted chemical probes. Our hypothesis is that spatial analysis will provide single cell-specific profiling to elucidate biological pathways and treatment effectiveness.