Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos

Camille Lombard-Banek, Sally A. Moody, M. Chiara Manzini, Peter Nemes

Research output: Contribution to journalArticlepeer-review

40 Scopus citations

Abstract

Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420 000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified ∼750-800 protein groups (<1% false-discovery rate) by analyzing just ∼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.

Original languageEnglish (US)
Pages (from-to)4797-4805
Number of pages9
JournalAnalytical Chemistry
Volume91
Issue number7
DOIs
StatePublished - Apr 2 2019
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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