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

Significance Statement

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.

About the author

Peter Nemes holds a PhD in Chemistry from the George Washington University (Washington, DC), where he developed laser ablation electrospray ionization mass spectrometry for in situ and in vivo analysis as well as molecular imaging in two and three dimensions as a PhD graduate student (advisor: Prof. Akos Vertes). He completed postdoctoral training in analytical neuroscience at the University of Illinois—Urbana-Champaign (mentor: Prof. Jonathan V. Sweedler), where he developed mass spectrometry technologies to measure small and large molecules in single neurons and to image their spatial distribution. One of these technologies was single-cell capillary electrophoresis, which revealed metabolomic heterogeneity between different neuron types in the central nervous system of Aplysia californica and adoptability of the single-neuronal metabolome to external conditions. Another technology was a custom-built MALDI-C60-SIMS dual ion source mass spectrometer that helped probe the spatial distribution of small-to-large molecules in single neurons. In 2011, Dr. Nemes joined the Food and Drug Administration (FDA, Silver Spring, MD) as a Principal Investigator, where he developed mass spectrometry-based technologies to enable the high-throughput screening of chemical contaminants in regulated drug products and medical devices. There, he developed a mass spectrometry facility and served as the Laboratory Leader of the Laboratory of Chemical Contamination at the Division of Chemistry and Materials Science. In 2013, Dr. Nemes became an Assistant Professor at the Department of Chemistry of the George Washington University. His research develops high-sensitivity mass spectrometry platforms to assess the spatiotemporal evolution of metabolic and proteomic processes. Current work in the Nemes laboratory elucidates molecular mechanisms by which (i) cells acquire different fates in the developing vertebrate embryo and the central nervous system and (ii) respond to external stimuli such as drugs of treatment and toxins. Prof. Nemes has authored 30 peer-reviewed publications, 6 book chapters, and 80+ presentations, and holds 4 licensed patents. He received the 2008 International Research Fellowship award by the Dimitris N. Chorafas Foundation (Luzern, Switzerland), the 2009 American Institute of Chemists prize in Chemistry by the American Institute of Chemists (Washington, DC), the 2010 Science and Technology Innovation Award by Baxter Healthcare Corporation (Chicago, IL), the 2011 Special recognition by the FDA (Silver Spring, MD), and the 2016 Arthur Findeis Award for Achievements by a Young Analytical Scientist by the American Chemistry Society. Prof. Nemes is a Beckman Young Investigator by the Arnold and Mabel Beckman Foundation.

About the author

Camille Lombard-Banek obtained a Master of Science in chemical engineering from the Ecole Nationale de Chime de Lille (Paris, France) and in chemistry from the University of Toledo (Toledo, OH). She is currently completing her PhD at the George Washington University (Washington, DC) under the tutelage of Prof. Peter Nemes. Her PhD research focuses on advancing mass spectrometry technology to enable the characterization of basic molecular processes in single embryonic cells as they commit to different tissue fates. Mrs. Lombard is recipient of the 2015 Georges Guiochon Student Travel Award from the Washington Chromatography Discussion Group (Washington, DC).

About the author

Sally A. Moody received her Ph. D. in Neuroscience from the University of Florida College of Medicine where she studied motor axon guidance cues in the trigeminal system of the chick embryo. She did her postdoctoral work in the Department of Neurobiology and Anatomy at the University of Utah School of Medicine where she studied axon guidance and cell lineage in Xenopus embryos. She was an Assistant and then tenured Associate Professor in the Department of Anatomy and Cell Biology at the University of Virginia School of Medicine, with a joint appointment in the Department of Neuroscience. There she made extensive fate maps of the cleavage stage Xenopus embryos, demonstrated the roles of various extracellular matrix proteins in axon growth, identified a maternal RNA contribution to neural fate and demonstrated lineage influences on the determination of amacrine cell fate. She moved to the George Washington University School of Medicine and Health Sciences in 1992, where she is Professor of Anatomy and Regenerative Medicine. Currently, her laboratory is studying the gene regulatory network that stabilizes neural stem cell fate and the factors that are required for cranial sensory placode development. She has served on the editorial boards of several journals in the fields of neuroscience, developmental and stem cell biology.

Journal Reference

Angew Chem Int Ed Engl. 2016 Feb;55(7):2454-8.

Lombard-Banek C¹, Moody SA², Nemes P³.

[expand title=”Show Affiliations”]

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].

[/expand]

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. 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.

Go To Angew Chem Int Ed Engl