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

描述（由申请人提供）：细胞凋亡和自噬细胞死亡是动物发育过程中发生的两种最主要的程序性细胞死亡形式。虽然我们对细胞凋亡的调控了解甚多，但对于细胞自噬死亡的机制，以及区分这些最突出的生理细胞杀伤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死亡的理想系统。大量的胚胎和唾液腺细胞使其能够从其他组织中快速纯化和分离，提供了为数不多的高级动物遗传系统之一，在该系统中，分子和生化方法可以用于研究发育生物体背景下纯化的死亡细胞群体。这些特性表明，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，并为研究其功能提供了一个很好的系统。程序性细胞死亡遗传途径在果蝇和人类之间是非常保守的，这表明对果蝇细胞死亡的研究将与高等生物直接相关。

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.