2019/03/03 by Camille Lombard‐Banek, Sally A. Moody, M. Chiara Manzini +1 · 3 citations
Chemistry · Engineering · #Advanced Proteomics Techniques and Applications #Biochemistry #Biology #Biosensors and Analytical Detection #Cell #Cell biology #Chemistry #Chromatography #Computational biology #Embryo #Gene #Mass Spectrometry Techniques and Applications #Mass spectrometry #Molecular biology #Proteome #Proteomics #Quantitative proteomics #Single-cell analysis #Xenopus #Zebrafish
paper · doi:10.1021/acs.analchem.9b00345
openalex publication_date 2019/03/03 · openalex created_date 2019/03/11 · openalex updated_date 2026/08/01
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.