Protein analysis at the single-cell level opens new possibilities to study cell-specific gene expression during embryonic development. Traditional mass spectrometry approaches average across large numbers of cells, which limits important information on cell-to-cell heterogeneity such as differential cell fates during embryonic development. We recently enabled detecting proteomic cell-to-cell differences in the developing vertebrate embryo by developing a single-cell bottom-up proteomics approach using a custom-built capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS). Here, we extend this technology to progressively smaller cells in later stages of the embryo by integrating in-situ microsampling with CE-ESI-MS. Briefly, we used a tapered microcapillary to collect ~10 nL from identified cells in the frog (Xenopus laevis) embryo. Sampled proteins were digested via a bottom-up proteomic workflow that we downscaled to the total protein content of the collected cell material. After evaluating various solvent mixtures to aid digestion, we successfully identified ~350 different protein groups on average from the micro-sampled cells. This compared favorably to the number of proteins identified from dissected whole cells (~250). Next, we used this approach to measure protein translation in smaller cells from later embryonic stages. We chose four different cell stages, 2 belonging to the cleavage stage (16- and 32-cell stages), and 2 belonging to the blastula stage (64- and 128-cell stages). These measurements allowed us to identify 470 protein groups and quantify ~450, 175 of which were quantifiable across all stages and all biological replicates. These measurements demonstrated differential protein production when compared to the 16-cell stage. Using unsupervised cluster analysis, we uncovered protein groups with characteristic temporal trends across the developmental stages. Our microanalytical single-cell proteomics approach opens new opportunities to understand how cell specific gene translation induces cell differentiation and organ development. We foresee this technology to be widely applicable to other models in cell and developmental biology.

In-situ single-cell proteomics in the frog embryo by bottom-up mass spectrometry

254th National Meeting (2017)

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