Development of Microanalytical Mass Spectrometry Tools to Probe Proteomic Differences between Single Embryonic Cells in Early Vertebrate Embryos

Measurement of proteins in single-embryonic cells can provide important information on how proteins contribute to determining cell fate and body patterning, raising the potential to understand the underlying causes of healthy and impaired embryonic development. However, to extend the measurements of proteins to single-cell technological advances are needed. In this dissertation, we describe how we developed mass spectrometry based approaches for the untargeted analysis of proteins in single embryonic cells. The research presented herein highlights the strategies developed based on capillary electrophoresis coupled to high-resolution mass spectrometry for the analysis of proteins. The developed tools enabled us to probe for chemical heterogeneity between single embryonic cells of early developing embryos. The results contributed new information to cell and developmental biology. Chapter 1 introduces the current state of proteomic analysis for cell and developmental biology, as well as our animal model for this study Chapter 2 describes how we developed a capillary electrophoresis electrospray ionization coupled to high-resolution mass spectrometry (CE-ESI-HRMS) for the analysis of single dissected neural fated cells.Chapter 3 presents further advances in our single-cell analysis workflow, with the introduction of multiplexing with tandem-mass-tags, enabling the analysis of three differently fated cells of the 16-cell X. laevis embryo.Chapter 4 discusses the development of a microprobe sampling approach allowing for the collection of protein content in live-embryos. This approach enabled for the first time temporal proteomic analysis of single-neural fated cell clones. Chapter 5 details the extension of our microprobe sampling approach for the combined metabolomics and proteomic inference in single embryonic cells. Chapter 6 summarizes the results from the previous chapter and reflects on some of the advances needed for proteomic analysis with single-cell resolution to move forward.

Author

• Lombard-Banek, Camille

Language

• en

Keyword

• Cell fate
• Single cell
• Capillary electrophoresis
• Embryonic development

Date created

• 2018

Type of Work

• Dissertation

License

• All rights reserved

Rights statement

• In Copyright

GW Unit

• Chemistry

Degree

• Ph.D.

Advisor

• Nemes, Peter

Committee Member(s)

• Massiah, Michael
• Marginean, Ioan
• Vertes, Akos
• Rodriguez, Erik

Persistent URL

•  https://scholarspace.library.gwu.edu/etd/xw42n818s