Single-Cell Metabolic Analysis and Molecular Imaging of Biological Tissues by Mass Spectrometry by Laith Samarah, GW Graduate Student, Vertes Lab

Laith Samarah, GW Department of Chemistry, Graduate Student, Vertes Lab

Characterizing the phenotypes of complex biological systems presents unique challenges marked by 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 analysis of compounds that describe the phenotypes of tissues and cells, respectively, including metabolites, lipids, peptides, and proteins. My projects utilized MSI and single-cell analysis to explore the molecular framework of the symbiosis between soybean (Glycine max) and the nitrogen-fixing soil bacteria, Bradyrhizobium japonicum. Using nanophotonic ionization from silicon nanopost arrays (NAPAs), ion intensities for several biooligomers were spatially mapped throughout root nodule tissue sections, revealing potential correlations between their functions and spatial distributions. The regulation of the biooligomer synthesis was investigated by comparing their abundances in nodules harboring genetically variant rhizobia. To explore the inherent cellular heterogeneity in root nodules, single infected cells were analyzed by fiber-based laser ablation electrospray ionization (f-LAESI) coupled to 21 Tesla Fourier transform ion cyclotron resonance (21T FT-ICR) MS. The ultrahigh mass resolution and dynamic range of the mass spectrometer were exploited to elucidate the elemental compositions for 20 metabolites by isotopic fine structures (IFSs) from a single cell.  Metabolic noise was determined for several compounds in infected cells, providing insights into potential correlations between the metabolite classes and the corresponding noise levels. Additional insight into cellular heterogeneity was gleaned by determining the metabolite abundance distributions over the studied cell population. To investigate the long-distance transport of metabolites between root nodules and other plant parts in-vivo, xylem sap was selectively sampled using capillary microsampling, and analyzed by electrospray ionization (ESI) MS. The abundance levels for specific nitrogen-containing compounds varied in the xylem sap of infected and uninfected plants, indicating that nitrogen transport was regulated depending on its source.