High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo

Abstract

Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of the basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in the 16-cell embryo of the South African clawed frog (Xenopus laevis) that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

“…In the past five years, great strides have been made to extend the application of MS-based proteomic to the measurement of single cells. This application was rendered possible by advances in sample preparation, enhanced nanoscale separation modalities discussed in Section 3.2, and improvement in MS instrumentation [106][107][108]. Single cell handling and sample preparation for proteomic analysis is perhaps the most challenging step of the workflow and requires more advanced technology than described in Section 3.1 to accommodate for the much-reduced protein material afforded by single cells.…”

New‐generation mass spectrometry expands the toolbox of cell and developmental biology

“…Quantitative approaches are used to determine relative or absolute levels of hundreds‐to‐thousands of these molecules. (Figures adapted with permission from references (Lombard‐Banek et al, ; Onjiko et al, ; Onjiko et al, ))…”

“…Additionally, these quantitative proteomic data uncovered gene translational heterogeneity between cells that occupy the animal‐vegetal and dorsal‐ventral axis of the embryo, which complements transcriptional cell heterogeneity only known along the animal‐vegetal axis (Flachsova, Sindelka, & Kubista, ). Furthermore, we have also adapted LFQ for single‐cell CE‐MS to find proteins that are produced at comparable and variable levels between neural‐fated blastomeres from different 16‐cell embryos (Lombard‐Banek et al, ). Single‐cell CE‐ESI‐MS has also allowed the detection of ∼250–350 different proteins in protein digests that estimate the protein content of a handful‐to‐single neurons in the mouse cerebral cortex (Choi et al, ; Nemes et al, ), raising a potential to study the formation of cell heterogeneity in the nervous system.…”

“…Over the last couple of years, MS sensitivity and quantification has been advanced to enable the characterization of gene translation in progressively smaller tissues and single cells by us (Choi, Zamarbide, Manzini, & Nemes, ; Lombard‐Banek et al, ; Lombard‐Banek, Moody, & Nemes, ; Lombard‐Banek, Lombard‐Banek, Reddy, Moody, & Nemes, ) and others (Smits et al, ; Sun et al, ; Virant‐Klun et al, ; Wuhr et al, ). Xenopus laevis has been a particularly powerful model to facilitate these technological advances by providing substantial amounts of proteins and considerably large cells with stereotyped tissue fates (Dale & Slack, ; Moody, ).…”

“…Quantitative approaches are used to determine relative or absolute levels of hundreds-to-thousands of these molecules. (Figures adapted with permission from references(Lombard-Banek et al, 2016b;Onjiko et al, 2015;Onjiko et al, 2016b)) hatched tadpole (NF 33). Quantification of 4,000 proteins between stages 1 and 22 using 2-dimensional nanoLC-MS and iTRAQ found dramatic changes in protein profiles during the mid-blastula transition in Xenopus laevis(Sun et al, 2014).…”