Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content

Abstract

Single cell analysis is required to understand cellular heterogeneity in biological systems. We propose that single cells (blastomeres) isolated from early stage invertebrate, amphibian, or fish embryos are ideal model systems for the development of technologies for single cell analysis. For these embryos, although cell cleavage is not exactly symmetric, the content per blastomere decreases roughly by half with each cell division, creating a geometric progression in cellular content. This progression forms a ladder of single-cell targets for the development of successively higher sensitivity instruments. In this manuscript, we performed bottom-up proteomics on single blastomeres isolated by microdissection from 2-, 4-, 8-, 16-, 32- and 50-cell Xenopus laevis (African clawed frog) embryos. Over 1,400 protein groups were identified in single-run reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry from single balstomeres isolated from a 16-cell embryo. When the mass of yolk-free proteins in single blastomeres decreased from ~0.8 μg (16-cell embryo) to ~0.2 μg (50-cell embryo), the number of protein group identifications declined from 1,466 to 644. Around 800 protein groups were quantified across four blastomeres isolated from a 16-cell embryo. By comparing the protein expression among different blastomeres, we observed that the blastomere-to-blastomere heterogeneity in 8-, 16-, 32- and 50-cell embryos increases with development stage, presumably due to cellular differentiation. These results suggest that comprehensive quantitative proteomics on single blastomeres isolated from these early stage embryos can provide valuable insights into cellular differentiation and organ development.

Transformative Opportunities for Single-Cell Proteomics

“…21–23 More recently, ESI−MS has been used in conjunction with other significant innovations to detect many more proteins in single human oocytes 24 or small samples from single embryonic frog cells (blastomeres). 25,26 Even with mammalian cells having diameter ∼15 μ m, ESI−MS has made progress in quantifying increasing number of proteins in relatively small number of cells, reaching thousands of proteins in cell lysates corresponding to hundreds of cells 27 or even fewer cells. 28 Recently, we reported a method that allows quantifying over a thousand proteins across many single mouse stem cells.…”

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry

“…Combined with continuous developments in HRMS technology for Xenopus , such as multiplexing quantification 23 and ultrasensitive detection, 26,53,54 the methodologies tested here afford possibilities to study developmental processes with progressively deeper molecular coverage, even at the level of single cells. 26,27,55 …”

Categorizing Cells on the Basis of their Chemical Profiles: Progress in Single-Cell Mass Spectrometry

“…30,42 Improvements in sheathless CE-MS interfaces have allowed investigation of complex bioanalytical problems, as in the characterization of protein isoforms and combinatorial PTMs reported by Yates and co-workers. 69,70 Recent examples from Dovichi 71 and Nemes 72,73 of the developing Xenopus laevis embryo demonstrate the great promise for CE-MS-based single-cell proteomics.…”