Metalloproteome of the Aging Rat Brain Mitochondria

描述（由申请人提供）：线粒体富含金属。需要精确水平的金属，并且通常与特定的同源蛋白结合以实现最佳的线粒体功能。然而，在衰老过程中，发生遗传和表观遗传变化，导致线粒体功能衰退，包括改变金属稳态。金属含量和金属蛋白水平的波动会导致亚细胞和细胞功能障碍，最终导致疾病。为了清楚地了解金属稳态和线粒体功能之间的关系，必须提供线粒体中所有金属（金属组）和所有金属蛋白（金属蛋白组）的完整描述和定量，但技术障碍阻碍了这一目标的进展。然而，最近开发了将超灵敏元素分析与高通量蛋白质组学工具相结合的新方法，从而能够分离和鉴定数百种蛋白质并同时揭示结合的金属。该技术最适合于量化金属和识别离散蛋白质组（如线粒体）中的金属结合蛋白。通过测量金属含量和金属蛋白表达模式随时间的变化模式，可以更全面地了解衰老对线粒体功能的影响。在这项初步研究中，（1）将对大鼠大脑皮层线粒体中的金属和金属蛋白含量进行定量，因为皮层区域是衰老和神经退行性疾病的已知靶点。然后，（2）通过绘制年轻、中年和老年大鼠大脑皮层金属含量和金属蛋白模式的主要变化图，验证线粒体金属稳态在衰老过程中发生变化的假设。将对通过天然液相等电聚焦随后连续洗脱电泳分离的线粒体蛋白组分进行表达和元素含量分析。分别用扫描透射离子显微镜和质子诱导X射线发射定量蛋白质质量和元素含量，然后结合MALDI-TOF和LC-TOF-MS分析确定的蛋白质鉴定，以生成大鼠皮质线粒体的金属组和金属蛋白质组。这些结果将作为一个可在因特网上搜索的数据库公布，突出显示与年龄有关的变化。因此，这项工作具有整合线粒体代谢和生理学的许多方面的重要潜力，并揭示了对抗衰老相关衰退的新机会。

DESCRIPTION (provided by applicant): Mitochondria are very metal rich. Precise levels of metals are required and typically bind to specific cognate proteins for optimal mitochondrial function. However during aging, genetic and epigentic changes occur that lead to decay in mitochondrial function, including altered metal homeostasis. Fluctuations in metal content and metalloprotein levels can cause subcellular and cellular dysfunction, and eventually disease. To clearly understand the relationship between metal homeostasis and mitochondrial function, a complete description and quantitation of all metals (the metallome) and all metalloproteins (the metalloproteome) in the mitochondria must be made available, yet technical obstacles have hindered progress towards this goal. Recently though, novel methods were developed that merge ultrasensitive elemental analysis with high- throughput proteomic tools resulting in the capacity to separate and identify hundreds of proteins and simultaneously reveal bound metals. This technology is optimally suited to quantify metals and identify metal-binding proteins within discrete proteomes, such as the mitochondria. By measuring the shifting patterns in metal content and metalloprotein expression patterns over time, a more comprehensive understanding of the effects of aging on mitochondrial function can be ascertained. In this preliminary study, (1) metal and metalloprotein content in mitochondria from the rat brain cortex will be quantitated, as cortical regions are a known target of aging and neurodegenerative disease. Then, (2) the hypothesis that mitochondrial metal homeostasis is altered during aging will be tested by mapping the major changes in metal content and metalloprotein patterns from young, middle-aged, and old adult rat cortex. Analysis of expression and elemental content will be performed on mitochondrial protein fractions separated by native liquid-phase isoelectric focusing followed by continuous elution electrophoresis. Protein mass and elemental content will be quantitated with scanning transmission ion microscopy and proton induced X-ray emission, respectively, and then combined with protein identification determined from MALDI-TOF and LC-TOF-MS analyses to generate the metallome and metalloproteome for rat cortical mitochondria. These results will be published as an Internet-based searchable database, highlighting age-related changes. Thus, this work has significant potential to integrate numerous aspects of mitochondrial metabolism and physiology and reveal novel opportunities for combating aging-related decline.