Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo

Abstract

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. 25amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20ng, 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. Back to the beginning: the cell. The encoded proteome offers basic insight into normal development. Capillary electrophoresis, electrospray ionization, high-resolution mass spectrometry, and bottom-up proteomics were integrated to enable the identification of 1709 proteins in single embryonic Xenopus cells. Quantification of hundreds of proteins revealed translational differences between cells that give rise to different tissues during development.