ABSTRACT: 10.1021/acs.analchem.6b01921
Article: Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content.
Project description:Tissues from the eye primordia, lateral endoderm, and posterior; neural plate of stage 15 Xenopus laevis embryos were isolated and normalized to stage 15 whole embryos. Three biological replicates were prepared for each tissue and the expression patterns were profiled using Affymetrix Xenopus Laevis GeneChip microarrays. Experiment Overall Design: Tissues from the eye primordia, lateral endoderm, and posterior Experiment Overall Design: neural plate of stage 15 Xenopus laevis embryos were isolated and normalized to stage 15 whole embryos. Three biological replicates were prepared for each tissue and the expression patterns were profiled using Affymetrix Xenopus Laevis GeneChip microarrays.
Project description:This project used a custom-built capillary electrophoresis (CE) mass spectrometry (MS) platform to demonstrate the scalable analysis of proteins from single cells of varying sizes and protein content in different vertebrate biological models. Neural tissue fated giant cells were identified in cleavage-stage Xenopus laevis (frog) embryos, and 18 ng from its protein content was analyzed. From cultured primary neurons from the mouse, diluted samples containing ~125 pg of protein digest was measured, estimating to a portion of the somal protein content. Compared to traditional nano-flow liquid chromatography (nanoLC) MS, CE-MS provided higher sensitivity and faster analysis. CE-ESI-HRMS offers a viable approach for scalable single-cell proteomics.
Project description:This project used a custom-built capillary electrophoresis (CE) mass spectrometry (MS) platform to demonstrate the scalable analysis of proteins from single cells of varying sizes and protein content in different vertebrate biological models. Neural tissue fated giant cells were identified in cleavage-stage Xenopus laevis (frog) embryos, and 18 ng from its protein content was analyzed. From cultured primary neurons from the mouse, diluted samples containing ~125 pg of protein digest was measured, estimating to a portion of the somal protein content. Compared to traditional nano-flow liquid chromatography (nanoLC) MS, CE-MS provided higher sensitivity and faster analysis. CE-ESI-HRMS offers a viable approach for scalable single-cell proteomics.
Project description:Tissues from the eye primordia, lateral endoderm, and posterior neural plate of stage 15 Xenopus laevis embryos were isolated and normalized to stage 15 whole embryos. Three biological replicates were prepared for each tissue and the expression patterns were profiled using Affymetrix Xenopus Laevis GeneChip microarrays. Keywords: Three way comparison of Embryonic Tissue Isolation
Project description:Xenopus laevis embryos were injected with mRNA for EFTFs at 2-cell stage. Animal caps collected at stage 9, cultured to the equivalent of stage 15 and RNA extracted. Four biological replicates of the EFTF-injected and GFP-injected (control) caps were used to profile transcript expression patterns using Affymetrix Xenopus Laevis GeneChip microarrays. Experiment Overall Design: Xenopus laevis embryos were injected with mRNA for EFTFs at 2-cell stage. Animal caps collected at stage 9, cultured to the equivalent of stage 15 and RNA extracted. Four biological replicates of the EFTF-injected and GFP-injected (control) caps were used to profile transcript expression patterns using Affymetrix Xenopus Laevis GeneChip microarrays.
Project description:In this work, we developed a microprobe capillary electrophoresis high-resolution mass spectrometry approach to identify proteins in single identified cells in live embryos of the South African clawed frog (Xenopus laevis) and also zebrafish. We used a pulled borosilicate capillary to aspirate a calibrated portion of the cell of interest. Proteins in the collected aspirate were detected and quantified via a bottom-up proteomic workflow that was downscaled to the single cells. The microprobe mass spectrometry platform is scalable to smaller cells in live, morphologically complex, developing embryos. As an example, we used this approach to analyze proteomic changes in cells that form a clone in the early developing Xenopus laevis embryo.
Project description:Xenopus laevis embryos were injected with mRNA for EFTFs at 2-cell stage. Animal caps collected at stage 9, cultured to the equivalent of stage 15 and RNA extracted. Four biological replicates of the EFTF-injected and GFP-injected (control) caps were used to profile transcript expression patterns using Affymetrix Xenopus Laevis GeneChip microarrays. Keywords: Two way comparison
Project description:We report a filter aided, single tip (FAST) method for ultrasensitive proteomic sample preparation. To minimize sample loss and contamination, the method reduces the surface area of the filter to ~0.1 mm2, the total volume of reagents to < 10 μL, and the number of sample transfer steps to two. 25,883 unique peptides and 3,069 protein groups were identified from 1,000 MCF-7 cells (~100 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (~200 ng yolk free protein/blastomere) generated 20,943 unique peptides and 2,597 protein groups; the proteomic profile clearly differentiated left and right blastomeres, and provides strong support for models in which this asymmetry is established early in the embryo.
Project description:This project integrated data-independent acquisition (DIA) with capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) to enable fast single-cell proteomics. With <15 min of effective separation time in a bottom-up proteomics setting, this technology returned ~1,200 proteins from a single HeLa-cell-equivalent proteome amount. Furthermore, using capillary microsampling to collect the proteome from identified cells in a cleavage-stage embryo of the vertebrate Xenopus laevis (frog) embryo, ~1,400 proteins were identified by measuring ~5 ng of the subcellular proteome content aspirated from live Xenopus embryos. CE-ESI-MS with DIA enhanced proteome detection sensitivity and improved analytical throughput.