CHARACTERIZATION OF THE EARLY APOPTOTIC MITOCHONDRIAL PROTEOME

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 哺乳动物和酵母线粒体的一些蛋白质组学研究已经确定了线粒体蛋白质组中超过1000种蛋白质的不断增长的列表。 各种研究之间的重叠限于60-80%，这些组分的相对丰度随组织类型而变化。 基于线粒体进化的内共生体模型（Gabaldon，2004），线粒体蛋白质的数量估计为2000，因此线粒体蛋白质组仍然不完整。 绝大多数线粒体蛋白是核编码的，并且预期含有用于线粒体靶向的N-末端肽信号。 有趣的是，在哺乳动物线粒体蛋白质组中，只有三分之一的蛋白质使用生物信息学分析预测含有信号肽基序，而另外三分之一很容易用已知的线粒体酶功能注释。 其余三分之一的样本是典型的功能不明确的新型线粒体定位蛋白，在一些研究中，这个数字超过总蛋白质列表的一半。 这些蛋白质列表中明显缺少凋亡调节蛋白，其在高度调节的细胞死亡途径中与线粒体膜相关。 该项目将解决1）使用早期凋亡信号的受控生物学和药理学诱导的线粒体蛋白质的功能注释和2）用于蛋白质靶向线粒体的N-末端肽的分析。 为了实现这些目标，我们正在研究完整线粒体的自由流电泳（FFE）纯化，减少高丰度核糖体和核组分的污染。 我们还使用化学生物学酶促方法用枯草杆菌酶标记线粒体蛋白的游离N-末端用于亲和捕获。 质谱设备在该项目中的作用是处理细胞器制备、肽测序和N-末端肽的生物信息学分析的所有方面。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Several proteomic studies of mammalian and yeast mitochondria have identified a growing list of over 1000 proteins in the mitochondrial proteome. The overlap between various studies is limited to 60-80% and the relative abundance of these components varies with tissue type. The number of mitochondrial proteins has been estimated at 2000, based on an endosymbiont model for mitochondrial evolution (Gabaldon, 2004), thus the mitochondrial proteome is still incomplete. The vast majority of mitochondrial proteins are nuclear encoded and are expected to contain N-terminal peptide signals used for mitochondrial targeting. Interestingly, only one third of the proteins in the mammalian mitochondrial proteome are predicted to contain a signal peptide motif using bioinformatic analysis, while another third are easily annotated with known mitochondrial enzymatic functions. The remaining third of the sample are typically novel mitochondrial localized proteins of ambiguous function, and in some studies this number is greater than half of the total protein list. Conspicuously absent from many of these protein lists are the apoptosis regulating proteins, which associate with mitochondrial membranes in a highly regulated pathway for cell death. This project will address 1) functional annotation of mitochondrial proteins using controlled biology and pharmacological induction of early apoptosis signaling and 2) analysis of N-terminal peptide for targeting of proteins to mitochondria. To accomplish these goals, we are investigating free flow electrophoresis (FFE) purification of intact mitochondria, reducing contamination by high abundance ribosome and nuclear components. We are also using a chemical biology enzymatic approach to label with subtiligase the free N-termini of mitochondrial proteins for affinity capture. The role of the Mass Spectrometry Facility in this project is to handle all aspects of the organelle preparation, peptide sequencing and bioinformatic analysis of N-terminal peptides.