Lombard-Banek C et al. (2016), High-Sensitivity Mass Spectrometry for Probing ...

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Front Cell Dev Biol 2016 Jun 24;4:100. doi: 10.3389/fcell.2016.00100.

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

Lombard-Banek C , Moody SA , Nemes P .

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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.

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	Figure 1. Analytical workflow for the bottom-up measurements of protein expression in single embryonic cells. A custom-built high-sensitivity capillary electrophoresis electrospray ionization mass spectrometer (CE-ESI-MS) is used to identify and quantify proteins.
	Figure 2. Isolation of identified cells and processing of their protein content. Example shows how the epidermal-fated ventral-animal cell (named V11) was identified in the 16-cell X. laevis embryo based on pigmentation, cell size, and location in reference to established cell fate maps (Moody, 1987a). The cell was processed via bottom-up proteomic workflow, and the resulting peptides collected for proteomic analysis. Key: DTT, dithiothreitol; IAD, iodoacetamide. Scale bar = 200 μm (embryo), 1.25 mm (vial).
	Figure 3. Schematics of the high-sensitivity proteomic analyzer. The platform integrates microanalytical capillary electrophoresis (CE), electrospray ionization (ESI), and high-resolution tandem mass spectrometry (HRMS2). Scale bar = 150 μm (ESI), 1.5 mm (CE panel). Key: HVPS, high-voltage power supply. Figure adapted with permission from Lombard-Banek et al. (2016a).
	Figure 4. Peptide identification/quantification in CE-ESI-HRMS2 using a bottom-up strategy. (A) Peptides are electrophoretically separated (left panel) and their accurate mass is measured (right panel). (B) Peptide signals are sequenced by tandem MS (MS2). For example, a signal was detected with m/z 572.33 at ~50 min separation, which was assigned to the sequence LGLGLELEA based on the MS2 data. (C) Peptides are quantified and assigned to the source protein. Tandem mass tags (TMT) with different m/z values (indicated by asterisks of different color in left panel) are used to barcode peptides from different cells, allowing their simultaneous analysis (multiplexing) with higher throughput (left panel). For example, the sequence LGLGLELEA was unique to the voltage-dependent anion channel 2 protein in the Xenopus proteome. The presence of other peptides allowed identifying this protein in high sequence coverage; see detected sequence in green (right panel).
	Figure 5. Examples of protein identification–quantification between single embryonic cells. (A) The D11, V11, and V21 cells have different tissues fates in the frog X. laevis. Scale bars: 250 μm. Figure reprinted with permission from Onjiko et al. (2015). (B) These cells were dissected from different 16-cell X. laevis embryos and analyzed using multiplexed (left panel) and label-free quantification (right panel). Volcano plots reveal gene translation differences between the V11, D11, and V21 cell types (left). Pearson correlation analysis of protein expression finds similar protein expression for the majority of proteins between D11 blastomeres, and detectable differences for others (right panel). Figures adapted with permission from Lombard-Banek et al. (2016a,b).

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Abrams, Early zebrafish development: it's in the maternal genes. 2009, Pubmed