Discovery (untargeted) characterization of the metabolome in single-cells as they undergo differentiation promises to elevate our understanding of molecular processes during normal and impaired development, but this requires next-generation mass spectrometry capable of exceptional sensitivity. Using thousands–millions of cells, mass spectrometry (MS) allows for unbiased and label-free detection of diverse types of metabolites. Here, we advance MS to enable, for the first time, the measurement of metabolites in single cells in live frog (Xenopus laevis) embryos. We combine capillary microsampling and a custom-built single-cell MS platform to enable the direct, in situ characterization of metabolites in multiple single cells within the same developing embryo. Furthermore, we used this technology to explore metabolic cell heterogeneity between adjacent dorsal and ventral cells in the 16-cell embryo, which are fated to give rise to distinct tissue types. We were able to aspirate ~10 nL portion of cellular content from each identified cell and then micro-extract metabolites from these limited samples. Cell extracts were measured using our custom-built volume-limited capillary electrophoresis electrospray ionization platform in conjunction with a time-of-flight mass spectrometer. A total of ~250 molecular features (unique m/z and migration time) were detected between cells, 55 of which were identified as small molecules (metabolites) based on accurate mass measurements, tandem MS, and migration time comparison to standards and databases. By conducting multivariate analysis (PCA, HCA, and PLSDA) on the resulting metadata, we found previously unknown metabolic concentration gradients between cells in the embryo. The technology presented in this work enabled the in situ characterization of metabolites among cells in the same vertebrate (frog) embryo and raises a potential to understand how metabolic changes contribute to cell differentiation during development of the vertebrate embryo or other animal models.

Single-cell metabolomics for tracking cell differentiation in the live embryo

254th National Meeting (2017)

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