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We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-μESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.
Figure 1. Microanalytical pipeline enabling multiplexing proteomic quantification of single embryonic cells in the 16âcell Xenopus embryo using microdissection, microâscale bottomâup proteomics, and a customâbuilt singleâcell CEâμESI platform for a highâresolution tandem mass spectrometer (HRMS2). Key: HVPS, high voltage power supply; Syr. Pump, syringe pump. Scale bars: 150â μm (embryo and μESI, leftâmiddle panels), 250â μm (microcentrifuge vial), 1.5â mm (separation, right panel).
Figure 2. Advancing bottomâup discovery proteomics to single cells using CEâμESIâHRMS. A)â Quantification curves for model peptides with 25âamol lower limit of detection and at least a 3âlogâorder linear dynamic range. B)â Evaluation of technical and biological repeatability across a week of measurements. C)â Proteomic coverage was enhanced using 20â ng digest by refining sample preparationâseparation (Steps 1â4), peptide sequencing (Steps 5â9), and data analysis (Steps 10â12). Experimental conditions are in Tableâ S2. D)â Comparing peptide identifications by CEâμESIâHRMS with nanoLCânanoESIâHRMS, the closest neighbor of bottomâup proteomic technology.
Figure 3. Singleâcell measurements uncovering translational asymmetry in the 16âcell Xenopus embryo. A)â Identification of 1709 different protein groups between D11, V11, and V21 cell types, suggesting proteomic cell differences (see peptide grouping in Figureâ S3 and proteins in Tableâ S3). B)â Gene ontology evaluation of biological processes (top) and subâcellular location of identified proteins (bottom). C)â Protein abundances covered a 5â6 logâorder dynamic range. Differential expression shown for Vdac2 (Inset). D)â Multiplexing quantification for 152 nonredundant protein groups between the D11/V11, D11/V21, and V11/V21 cell types. The volcano plot marks statistical and biological significance (p<0.05, â¥1.3âfold change) and labels select proteins. Significant protein differences are shown in Figureâ S4 and listed in Tableâ S5.
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