In situ metabolic characterization of single embryonic cells has potential to deepen molecular insights to cell differentiation and specialization. This demands specialized analytical technologies that ensure a broad coverage of the metabolome despite the minute cellular material afforded. Dissection of individual whole-cells and capillary electrophoresis-mass spectrometry (CE-MS) enabled us to recently uncover metabolic heterogeneity across the three key developmental axes of the embryo and to identify metabolites that alter tissue fates in Xenopus laevis, an established vertebrate model for cell and developmental biological studies. However, dissection is laborious at later stages of development, when cells are smaller. To enable the measurement of these smaller cells, we here developed and validated a microprobe single-cell CE-MS approach. The microprobe features a fabricated micropipette that enables the fast aspiration of ~10 nL of an identified single cell in the live embryo. Compounds in the cellular metabolic extract are electrophoretically separated and then ionized by our custom-built sheath-flow CE electrospray ionization interface. The ions are detected using a high-resolution tandem mass spectrometer. Despite miniscule volumes collected by this approach (~8% of the total cell volume), microprobe sampling yielded comparable number of ~250 molecular features to whole-cell dissection. We ascribe higher sensitivity to reduced ionization of spectral interference from abundant salts in the culturing media. We also used this microprobe strategy to inquire how the metabolome evolves spatiotemporally as the neural-tissue fated dorsal-animal cell undergoes two consecutive cell divisions (8–32-cell). Multivariate and statistical analysis of the spatiotemporal metadata revealed unique metabolic trends already at these early cleaving stages, suggesting metabolome reorganization as the embryonic cells continue to specialize to different tissue fates. Our work addresses an analytical challenge in single-cell metabolic characterization of live dividing cells. We anticipate that the developed approach is adaptable to other cell types and models.

Microprobe-CE-MS for in situ tracking of metabolome evolution in single-cells of the developing frog embryo

254th National Meeting (2017)

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