Technology for single cell proteomics and its application to the analysis of single Xenopus blastomeres

单细胞蛋白质组学技术及其在非洲爪蟾单个卵裂球分析中的应用

• 批准号: 9198018
• 负责人: Norman J Dovichi
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9198018
• 关键词: Address; Adult; Alkylation; Bioinformatics; Biological; Biological Models; Biology; Biopsy; Blood capillaries; Brain; Capillary Electrophoresis; Cell Lineage; Cell Separation; Cell Size; Cell division; Cells; Clinical; Communities; Complex; Computer software; Coupled; Cytolysis; Data; Data Analyses; Detection; Development; Developmental Biology; Devices; Digestion; Electrophoresis; Embryo; Ensure; Evaluation; Evolution; Fine-needle biopsy; Funding; Generations; Grant; High Pressure Liquid Chromatography; Label; Lasers; Manuals; Mass Chromatography; Medicine; Micrometastasis; Microtomy; Modeling; Monitor; Needles; Neoplasm Circulating Cells; Nervous system structure; Organ; Organism; Paper; Pathway interactions; Peptides; Phase; Preparation; Process; Proteins; Proteomics; Publications; Publishing; Pump; Reporting; Resources; Retina; Sampling; Series; Solid; Somatic Cell; Spinal Cord; System; Technology; Tissues; Trypsin; Tube; Xenopus; Xenopus laevis; blastomere structure; capillary; design; embryo stage 2; experience; experimental study; hatching; improved; insight; mass spectrometer; nano-electrospray; nervous system development; protein expression; public health relevance; single cell analysis; single cell proteins; single cell technology; tandem mass spectrometry; tool; zygote

 DESCRIPTION (provided by applicant): This proposal is a competitive renewal. In the 3.5 years since we received funding, we have published 29 papers acknowledging support from this grant; another five papers are under review. These publications address issues in diagonal capillary electrophoresis, including reactor design and improved capillary zone electrophoresis separations. We and others have demonstrated that capillary zone electrophoresis consistently provides more protein and peptide identifications than high performance liquid chromatography for mass-limited samples. We will take advantage of our experience to develop and evaluate technology for the bottom-up proteomic analysis of single blastomeres. Our first aim will develop on-column sample preconcentration technology, coupled with capillary zone electrophoresis peptide separation and an electrokinetically-pumped sheath flow nanoelectrospray interface with tandem mass spectrometry detection. Our second aim will develop on-column cell lysis, reduction and alkylation, digestion, and preconcentration in an integrated device, which will be evaluated using blastomeres along the D1 lineage. Our third aim provides bioinformatic support and ensures wide distribution of our results. This project also builds off of a recent publication from this team, which reported the first quantitative proteomic analysis of a developing embryo (Sci Rep. 2014; 4: 4365. PMID: 24626130). We studied single Xenopus laevis embryos at six stages of development, including stage 1 embryos. These stage 1 embryos have not divided, and constitute a single cell; we quantified the expression of 4,000 proteins from these single cells. We will extend our Sci Rep publication by quantitatively monitoring protein expression in single blastomeres from stage 2 of development through stage 20. These cells contain from 50-µg to 50-pg of protein, and form a natural progression of cells with progressively smaller protein content, ultimately ending at the size of a typical mammalian somatic cell. This progression of cell sizes is ideal for the development and evaluation of technology for single cell analysis. We will focus on the analysis of single cells isolated from the lineage of the blastomere D1, which ultimately form portions of the adult retina, spinal cord, and brain. Proteomic analysis of single blastomeres isolated from this lineage will provide insight into the assembly of the organism's nervous system.

 描述（由适用提供）：该提案是竞争性的续订。自从获得资金以来的3。5年中，我们发表了29篇认可该赠款的支持的论文；另外五篇论文正在审查中。这些出版物涉及对角毛细管电泳的问题，包括反应堆设计和改进的毛细管电泳分离。我们和其他人已经证明，毛细管区电泳始终提供的蛋白质和肽鉴定比质量有限的样品的高性能液相色谱法。我们将利用我们的经验来开发和评估技术的自下而上蛋白质组学分析的技术。我们的第一个目标将开发柱面样品预浓缩技术，再加上毛细管区电泳肽分离和电动倾斜的护套流量纳米电喷雾界面，并具有串联质谱检测。我们的第二个目标将在集成装置中发展出柱裂解，还原和酗酒，消化和预浓缩，将使用沿D1谱系的胚泡进行评估。我们的第三个目标提供了生物信息学的支持，并确保了我们的结果的广泛分配。该项目还建立在该团队的最新出版物中，该团队报告了开发胚胎的首次定量蛋白质组学分析（SciRep。2014; 4：4365。PMID：24626130）。我们在六个发育阶段（包括1阶段的胚胎）研究了单爪蟾laevis胚胎。这些第1阶段的胚胎尚未分裂，构成一个单元。我们量化了这些单个细胞的4,000种蛋白质的表达。我们将通过定量监测从发育到第20阶段的第2阶段的单个胚泡中的蛋白质表达来扩展SCI REP出版物。这些细胞含有从50 µg到50-Pg的蛋白质，并形成具有较小蛋白质的细胞的自然进展 内容，最终以典型的哺乳动物体细胞的大小结束。细胞尺寸的这种进展是用于开发和评估单细胞分析的理想选择。我们将重点介绍从胚泡D1谱系分离的单个细胞的分析，该细胞最终构成了成年视网膜，脊髓和大脑的一部分。从该谱系分离的单个胚泡的蛋白质组学分析将为有机体神经系统的组装提供见解。