Single Cell Neurometabolomics

Abstract

Metabolomics, the characterization of metabolites and their changes within biological systems, has seen great technological and methodological progress over the past decade. Most metabolomic experiments involve the characterization of the small-molecule content of fluids or tissue homogenates. While these microliter and larger volume metabolomic measurements can characterize hundreds to thousands of compounds, the coverage of molecular content decreases as sample sizes are reduced to the nanoliter and even to the picoliter volume range. Recent progress has enabled the ability to characterize the major molecules found within specific individual cells. Especially within the brain, a myriad of cell types are colocalized, and oftentimes only a subset of these cells undergo changes in both healthy and pathological states. Here we highlight recent progress in mass spectrometry-based approaches used for single cell metabolomics, emphasizing their application to neuroscience research. Single cell studies can be directed to measuring differences between members of populations of similar cells (e.g., oligodendrocytes), as well as characterizing differences between cell types (e.g., neurons and astrocytes), and are especially useful for measuring changes occurring during different behavior states, exposure to diets and drugs, neuronal activity, and disease. When combined with other omics approaches such as transcriptomics, and with morphological and physiological measurements, single cell metabolomics aids fundamental neurochemical studies, has great potential in pharmaceutical development, and should improve the diagnosis and treatment of brain diseases.

Keywords: Metabolomics; capillary electrophoresis; mass spectrometry imaging; matrix assisted laser desorption/ionization; neurochemistry; single cell measurements.

Figure 1

(a) Fabrication steps and structure of a Single-probe for ESI-MS; (b) photograph of a working Single-probe; (c) 40× magnification of the Single-probe tip with measurements obtained from the calibrated digital microscope; (d) setup schematic of an in situ real-time single cell MS analysis. Reproduced with permission from Pan, N., Rao, W., Kothapalli, N. R., Liu, R., Burgett, A. W. G., and Yang, Z. (2014) The single-probe: A miniaturized multifunctional device for single cell mass spectrometry analysis, Anal. Chem. 86, 9376–9380 (ref.). Copyright 2014, American Chemical Society.

Figure 2

Sequential analysis of the same individual cell with two separate MS systems. Once a cell has been located in the optical image (top), its location remains fixed through multiple analyses, allowing two instruments to probe the same set of selected cells. (A) MALDI-TOF MS (middle) of a rat cerebellum-derived cell followed by MALDI-FT-ICR MS (bottom). MALDI-TOF MS provides high throughput screening of thousands of cells to highlight rare or representative individuals. Here, FT-ICR MS provides high mass resolution and high mass accuracy for unequivocal elemental composition of selected cellular contents. (B) SIMS profiling (middle) followed by MALDI-TOF MS (bottom) with a DHB-coated, SCN-derived cell. SIMS provides information on small molecule compounds while MALDI-TOF MS effectively detects larger species, such as lipid dimers and peptides. The insets demonstrate overlap of intact lipid coverage from each modality. Reproduced with permission from Comi, T. J., Neumann, E. K., Do, T. D., Sweedler, J. V.: microMS: A python platform for image-guided mass spectrometry profiling. J. Am. Soc. Mass Spectrom. 28, 1919–1928 (2017) (ref.). Copyright American Society for Mass Spectrometry 2017, with permission from Springer.

Figure 3

Distribution of metabolites in cultured A. californica buccal neurons was performed by C₆₀-SIMS imaging. Cells were cultured on silicon tiles and stabilized with glycerol. C₆₀-SIMS ion images revealed that phosphocholine (PC) and α-tocopherol showed different locations in neurons. Cell bodies and processes are apparent in the PC image (m/z 184.08, 0–200 counts), while α-tocopherol accumulated almost exclusively within the cell bodies (m/z 430.39, 0–50 counts). Adapted with permission from Lanni, E. J., Dunham, S. J., Nemes, P., Rubakhin, S. S., and Sweedler, J. V. (2014) Biomolecular imaging with a C60-SIMS/MALDI dual ion source hybrid mass spectrometer: Instrumentation, matrix enhancement, and single cell analysis, J. Am. Soc. Mass Spectrom. 25, 1897–1907 (ref.). Copyright 2014, American Society for Mass Spectrometry, with permission from Springer.