Unraveling Cell Death via Top-Down/Bottom-Up Proteomics

描述(申请人提供)：细胞凋亡和自噬细胞死亡是发生在动物发育过程中的两种最突出的细胞程序性死亡形式。虽然人们对细胞凋亡的调控知之甚少，但对自噬细胞死亡的机制以及这些最突出的生理性细胞死亡形式的机制和生化变化知之甚少。果蝇胚胎和唾液腺是研究细胞凋亡和自噬程序性细胞死亡的理想系统。大量的胚胎和唾液腺细胞能够快速纯化并从其他组织中分离出来，这提供了为数不多的高级动物遗传系统之一，在这种系统中，分子和生化方法可以在发育中的有机体的背景下研究死亡细胞的纯化群体。这些属性表明，对果蝇细胞凋亡和自噬细胞死亡的研究将导致新的细胞死亡蛋白的鉴定，并为研究它们的功能提供一个很好的系统。果蝇和人类之间的程序性细胞死亡遗传途径是极其保守的，这表明果蝇细胞死亡的研究将直接与高等生物相关。 我们的研究目标是开发、优化和应用创新的蛋白质组技术，以全面分析细胞程序性死亡过程中的蛋白质谱。通过以果蝇黑腹果蝇为模型系统，这些蛋白质组研究将探索途径，并识别与发育中动物细胞死亡过程中Caspase激活相关的生物标记物。这一挑战将通过开发和应用基于毛细管的集成的自上而下/自下而上的多维分离平台来解决，该平台能够对完整蛋白质进行超高分辨率，随后将对大小可分辨的蛋白质进行原位蛋白分解消化，高通量多肽分离，以及超灵敏的多肽/蛋白质鉴定。结合果蝇遗传学和拟议的创新生物分析技术，可以在全基因组水平上研究细胞程序性死亡的调控，包括在全球范围内的蛋白质表达。

DESCRIPTION (provided by applicant): Apoptosis and autophagic cell death are the two most prominent forms of programmed cell death that occur during animal development. While much is known about the regulation of apoptosis, relatively little is known about the mechanisms underlying autophagic cell death, and what mechanism and biochemical changes distinguish these most prominent forms of physiological cell killing. Drosophila embryos and salivary glands are an ideal system to study apoptosis and autophagic programmed cell death. The mass of embryos and salivary gland cells enable rapid purification and separation from other tissues, providing one of the few higher animal genetic systems where molecular and biochemical approaches can be used to study a purified population of dying cells in the context of a developing organism. These attributes argue that studies of Drosophila apoptosis and autophagic cell death will lead to the identification of new cell death proteins, and provide an excellent system to study their function. The programmed cell death genetic pathway is extremely conserved between fruit flies and humans, indicating that studies of cell death in Drosophila will be directly relevant to higher organisms. Our research goal is to develop, optimize, and apply innovative proteome technology for the comprehensive analysis of protein profiles during programmed cell death. By using the fruit fly Drosophila melanogaster as a model system, these proteome studies will explore pathways and identify biomarkers associated with Caspase activation during cell death in developing animals. This challenge will be addressed through the development and application of a capillary-based and integrated top-down/bottom-up multidimensional separation platform capable of ultrahigh resolution of intact proteins, followed by in situ proteolytic digestion of size-resolved proteins, high throughput peptide separation, and ultrasensitive peptide/protein identification. The combined strength of Drosophila genetics and the proposed innovative bioanalytical technologies enables investigation into the regulation of programmed cell death at the genome-wide level including the expression of proteins on a global scale.