Single-cell mass spectrometry provides unique investigative opportunities to decipher cell-to-cell differences by extending discovery (untargeted) molecular measurements to limited amounts of materials. In this original work, Prof. Peter Nemes (PI) and Mrs. Camille Lombard-Banek of the GW Department of Chemistry enhanced the detection sensitivity of high-resolution mass spectrometry to ask with Prof. Sally A. Moody from the GW Department of Anatomy and Regenerative Biology how embryonic cells that give rise to different types of tissues host different proteomes. The research group developed workflows to dissect single embryonic cells from the 16-cell frog (Xenopus laevis) embryo, lyse and extract the proteins from these cells, and digest the proteins for analysis via a bottom-up proteomic workflow. The resulting peptides were bar-coded for cell identity using designer mass tags, pooled, and analyzed using a custom-built single-cell capillary electrophoresis electrospray ionization mass spectrometer. Quantification of ~150 protein groups revealed biologically and statistically significant differences in protein composition between embryonic cells that occupy the animal-vegetal and dorso-ventral axes of the embryo, forming nervous, skin, and gut tissues later during development of the vertebrate embryo. Besides demonstrating sufficient analytical sensitivity to use mass spectrometry for discovery measurements on single cells, the study provided new data for cell and developmental biology on the patterning of the early embryo. In combination with already available data on gene transcription, singe-cell mass spectrometry raises a powerful new opportunity to help better understand the molecular players underlying normal embryonic development. This research was funded by the National Science Foundation Division of Biological Infrastructure (grant no. 1455474).
Figure Legend: Quantification of proteomic differences between single embryonic cells in the 16-cell Xenopus embryo using proteomic single-cell high-resolution mass spectrometry.
- Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, The George Washington University, 800 22nd Street, NW, Suite 4000, Washington, DC, 20052, USA.
- Department of Anatomy and Regenerative Biology, The George Washington University, Washington, DC, 20052, USA.
- Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, The George Washington University, 800 22nd Street, NW, Suite 4000, Washington, DC, 20052, USA. [email protected]
We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog(Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.
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