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

 描述(申请人提供)：本建议书为竞争性续签。在我们获得资助的3.5年里，我们发表了29篇论文，确认得到了这笔赠款的支持；另有5篇论文正在审查中。这些出版物涉及对角线毛细管电泳法中的问题，包括反应器设计和改进的毛细管区带电泳法分离。我们和其他人已经证明，对于质量有限的样品，毛细管区带电泳始终比高效液相色谱提供更多的蛋白质和肽鉴定。我们将利用我们的经验开发和评估自下而上对单个卵裂球进行蛋白质组分析的技术。我们的第一个目标是开发柱上样品预富集技术，结合毛细管区带电泳肽分离和电动泵浦鞘流纳米电喷雾界面与串联质谱仪检测。我们的第二个目标将在一个集成设备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，并确保我们的结果得到广泛传播。该项目还建立在该团队最近的一份出版物的基础上，该团队报告了对发育中的胚胎进行的第一次定量蛋白质组分析(科学报告。2014；4：4365)。PMID：24626130)。我们研究了非洲爪哇6个发育阶段的单个胚胎，包括阶段1胚胎。这些阶段1的胚胎没有分裂，并构成了一个单细胞；我们量化了这些单细胞中4000个蛋白质的表达。我们将通过定量监测单个卵裂球从发育阶段2到阶段20的蛋白质表达来扩展我们的Sci Rep出版物。这些细胞含有50微克到50微克的蛋白质，并形成蛋白质逐渐变小的细胞的自然进化 内容，最终以典型哺乳动物体细胞的大小结束。这种细胞大小的发展是开发和评估单细胞分析技术的理想选择。我们将重点分析从卵裂球D1谱系中分离出来的单个细胞，这些细胞最终形成成人视网膜、脊髓和大脑的一部分。从这个谱系中分离出的单个卵裂球的蛋白质组学分析将为了解有机体神经系统的组装提供洞察力。

 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.