Single-Cell Metabolic Analysis and Molecular Imaging of Biological Tissues by Mass Spectrometry
Open AccessCharacterizing the phenotypes of complex biological systems presents unique challenges including the analysis of a dynamic network of metabolites with diverse chemical and physical properties and regulated over a wide range of concentrations. In recent years, mass spectrometry imaging (MSI) and single-cell analysis have played increasingly important roles in the biomolecular characterization of metabolites, lipids, peptides, and proteins in tissues and cells, respectively. This dissertation describes my efforts in advancing mass spectrometry (MS)-based techniques for single-cell metabolic analysis and implementing matrix-free MSI by laser desorption ionization (LDI) from nanophotonic silicon nanopost arrays (NAPA) to explore the molecular complexity of the legume-rhizobium symbiotic system in soybean (Glycine max) root nodules.Chapter 1 introduces the two analytical areas that are at the center of this work, MSI and single-cell MS. Spatial and chemical aberrations that distort the accuracy of information in MSI are described. The focus of MSI is given to techniques based on LDI from inorganic and nanophotonic platforms. A broad overview on single-cell MS and its evolving role in characterizing cellular heterogeneity is also given. Chapter 2 describes MSI of biooligomers in tissue sections from G. max root nodules using nanophotonic ionization from NAPA. Both spatial and size distributions for polyhydroxybutyrate (PHB) oligomers were characterized throughout the tissue sections by NAPA-MSI. Advantages for imaging these biooligomers using NAPA are evaluated in comparison with a mainstream MSI method, matrix-assisted laser desorption ionization (MALDI). Insights on the metabolism of different oligomers are revealed by comparing their relative abundances and spatial distributions in tissues harboring genetically-modified rhizobia (Bradyrhizobium japonicum). In Chapter 3, the focus in characterizing the molecular complexity of root nodules is shifted to single-cell analysis. Special challenges are mitigated by analyzing individual cells using fiber-based laser ablation electrospray ionization (f-LAESI) combined with 21 Tesla Fourier transform ion cyclotron resonance (FT-ICR) MS. The ultrahigh mass resolution and wide dynamic range of the 21T FT-ICR mass spectrometer were exploited to unravel the elemental compositions of up to 47 compounds by isotopic fine structures (IFS) from single cells. Direct ambient single-cell analysis by f-LAESI-21T FTICR-MS enabled the characterization of cellular heterogeneity through post-hoc identification of distinct subpopulations and metabolic noise measurements. Chapter 4 describes in-vivo analysis of long-distance metabolite transport and its regulation in G. max using direct whole sap electrospray ionization (ESI) MS and capillary microsampling ESI-MS. The chemical compositions of cells and the sap in the vasculature are analyzed and compared by capillary microsampling ESI-MS. The regulation of nitrogen transport in infected and uninfected plants is also evaluated. Chapter 5 gives a summary of the work presented in this dissertation and provides a condensed perspective on the future landscape for matrix-free MSI and single-cell MS, with potential breakthroughs and analytical challenges.
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