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

 描述（由申请人提供）：此提案是一个竞争性的更新。在获得资助后的3.5年里，我们发表了29篇论文，感谢这笔赠款的支持;另有5篇论文正在审查中。这些出版物解决了对角毛细管电泳中的问题，包括反应器设计和改进的毛细管区带电泳分离。我们和其他人已经证明，毛细管区带电泳始终提供更多的蛋白质和肽的鉴定比高效液相色谱质量有限的样品。我们将利用我们的经验，开发和评估技术的自下而上的蛋白质组分析单卵裂球。我们的第一个目标是开发柱上样品预浓缩技术，再加上毛细管区带电泳肽分离和电动泵鞘流纳米电喷雾接口与串联质谱检测。我们的第二个目标是在一个集成的装置中开发柱上细胞裂解、还原和烷基化、消化和预浓缩，这将使用沿着D1谱系的卵裂球进行评估。我们的第三个目标是提供生物信息学支持，并确保我们的结果广泛传播。该项目还建立在该团队最近发表的一篇论文的基础上，该论文首次对发育中的胚胎进行了定量蛋白质组学分析（Sci Rep. 2014; 4：4365）。PMID：24626130）。我们研究了单个非洲爪蟾胚胎在六个阶段的发展，包括第1阶段的胚胎。这些第1阶段的胚胎还没有分裂，并构成一个单细胞;我们定量了这些单细胞中4,000种蛋白质的表达。我们将通过定量监测从发育第2阶段到第20阶段的单个卵裂球中的蛋白质表达来扩展我们的Sci Rep出版物。这些细胞含有50-µg至50-pg的蛋白质，并形成具有逐渐变小的蛋白质的细胞的自然进展 内容，最终终止于典型哺乳动物体细胞的大小。细胞大小的这种进展对于单细胞分析技术的开发和评估是理想的。我们将重点分析从卵裂球D1谱系中分离的单细胞，这些细胞最终形成成人视网膜、脊髓和大脑的一部分。从这个谱系分离的单个卵裂球的蛋白质组学分析将提供对生物体神经系统组装的深入了解。

 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.