Technologies Trageting Mitochondral Proteome

描述(申请人提供)：线粒体在细胞代谢中具有多种基本职责，例如，通过有氧细胞的氧化磷酸化产生ATP，以及启动导致细胞凋亡的信号级联。线粒体蛋白质组的改变和线粒体功能的改变与多种退行性疾病、心脏病、衰老和癌症有关。由于线粒体是内源性产生的活性氧物种(ROS)的主要来源，因此它们被认为在衰老中发挥重要作用也就不足为奇了。衰老的自由基理论预测，线粒体的氧化损伤可以导致放大效应，受损的线粒体释放更多的ROS，进一步增加氧化损伤。考虑到大多数细胞类型和组织的渐进性年龄相关性功能变化，准确的年龄相关性线粒体蛋白质组是必要的，并将有助于阐明年龄相关疾病过程的分子机制和途径。为了大大提高线粒体蛋白质组的覆盖率，特别是在翻译后修饰(PTM)的识别方面，该项目旨在开发和展示一个基于毛细管凝胶电泳(CGE)的多维蛋白质分离平台，能够在质谱分析之前提供显著的分析物浓度和极高的分辨率来处理复杂的蛋白质混合物。与传统的基于SDS-PAGE的蛋白质组学方法相比，所提出的CGE/纳米反相液相色谱蛋白质组学技术将高度自动化，并为自上而下的蛋白质组学提供可靠和高通量的完整蛋白质分级，同时避免在一个集成平台中分析物的稀释和损失。通过与纳米级的胰酶膜反应器相结合，对基于CGE的分离和洗脱的蛋白质进行超快的蛋白水解性消化，使得自上而下/自下而上相结合的小鼠肝线粒体PTM的表征成为本研究中的模型系统。

DESCRIPTION (provided by applicant): Mitochondria have a variety of essential responsibilities in cellular metabolism, e.g. the production of ATP through oxidative phosphorylation in aerobic cells as well as the initiation of the signal cascade leading to apoptosis. Alteration of the mitochondrial proteome and altered mitochondrial function have been implicated in a variety of degenerative diseases, heart disease, aging, and cancer. Because the mitochondria are a major source of endogenously generated reactive oxygen species (ROS), it is not surprising that they have been proposed to play a major role in aging. The free radical theory of aging predicts that oxidative damage to the mitochondria can lead to an amplifying effect whereby damaged mitochondria release more ROS, further increasing oxidative damage. Considering the progressive age-dependent functional changes of most cell types and tissues, an accurate profile of age-dependent mitochondria proteome is mandatory and will shed light on the molecular mechanisms and pathways of age-associated disease processes. To greatly increase the mitochondria proteome coverage, particularly toward the identification of post-translational modifications (PTMs), this project aims to develop and demonstrate a capillary gel electrophoresis (CGE)-based multidimensional protein separation platform, capable of providing significant analyte concentration and extremely high resolving power for handling complex protein mixtures prior to mass spectrometry detection. In comparison with conventional SDS-PAGE-based proteome approaches, the proposed CGE/nano-reversed-phase liquid chromatography proteome technology will be highly automated and offer robust and high throughput fractionation of whole proteins for top-down proteomics while avoiding analyte dilution and loss in an integrated platform. By coupling with a nanoscale trypsin membrane reactor, the ultrafast proteolytic digestion of proteins resolved and eluted from the CGE-based separations enables the combined top-down/bottom-up characterization of PTMs in mouse liver mitochondria as the model system in this study.