Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics- Renewal

项目摘要 在每个生物体内，蛋白质都在工作，进行影响细胞功能的各个方面的活动。 实现如此广泛的蛋白质功能的一个关键因素是翻译后修饰 而产生大量功能蛋白质状态的序列修饰形成了单个基因 产品。这样的蛋白形式进一步直接偶联到所产生的生物分子的3D结构，这 进一步被招募到一系列动态的多蛋白质机器中。直接评估的结构 这些组装状态以及它们所包含的蛋白质形式对于理解人类至关重要 疾病。尽管如此，大多数结构仍然未知，对当前的技术来说是难以实现的，而且它们的 蛋白质形式的恭维仍然不透明。标准结构生物学方法(X射线、核磁共振和低温电子显微镜)， 虽然非常成功，但需要大量纯样品，精心优化才能生产 单分散蛋白质种群的各个方面，并消除光谱背景。此外，暂时性的 存在于复杂混合物中的多分散组件不能被分析。质谱仪(MS) 为解决这一具有挑战性的问题而开发的方法可以克服其中许多障碍。而这些 工具正在经历快速开发阶段，它们目前缺乏谨慎地评估 蛋白质形式对多蛋白组织的影响。因此，需要开发改进的MS 能够同时评估结构蛋白质组的方法，使蛋白质形式之间的联系成为可能 组成和三维结构为主体的动态、非均相的透明大分子络合物 生物医学的重要性。 此续订申请寻求构建新的、创新的MS技术，1)利用新的类别 化学标记试剂和混合标记方法，以促进完整序列的全面测序 多蛋白质复合体，2)利用下一代环离子迁移率-质谱仪(IM-MS) 产生高清晰度碰撞诱导展开(CIU)和自然自顶向下测序的技术 能够改进组装体内蛋白质形式识别的方法，3)产生了用于 膜蛋白复合体的直接测序，包括基于激光的洗涤剂簇的激活 为了提高数据质量，4)将电子捕获解离(ECD)与CIU相结合，用于即时注释 指纹数据，以及5)与原生生物兼容的蛋白质衍生化和清理的自动化方法 蛋白质组学。这项技术将被用于发现一系列精选的 蛋白质形式和复合体，每一种都与人类疾病有关。

Project Summary Within each organism proteins are at work carrying out activities which impact every aspect of cellular function. A key factor in achieving such a wide range of protein functions involves the post translational modifications and sequence modifications that act to produce a vast array of functional protein states form a single gene product. Such proteoforms are further coupled directly to the 3D structures of the biomolecules created, which are further recruited into a wide array of dynamic multi-protein machines. Directly assessing the structures of these assembly states, along with the proteoforms that they contain is crucial for understanding human disease. Despite this, most structures remain unknown and are refractory to current technologies, and their proteoform compliment remains opaque. Standard structural biology approaches (X-ray, NMR, and Cryo-EM), while highly successful, require pure samples in large quantities, painstakingly optimized to produce monodisperse protein populations in every respect, and to remove spectral background. Furthermore, transient and polydisperse assemblies that exist within complex mixtures cannot be analyzed. Mass spectrometry (MS) approaches developed to attack this challenging problem can overcome many of these obstacles. While these tools are undergoing a rapid development phase, they currently lack the ability to discreetly assess the influence of proteoforms on multiprotein organization. Consequently there is a need to develop improved MS approaches capable of simultaneously assessing the structural proteome, enabling links between proteoform composition and 3D structure for the host of dynamic, heterogeneous macromolecular complexes of clear biomedical importance. This renewal application seeks to construct new, innovative MS techniques that 1) utilize new classes of chemical tagging reagents and mixed tagging methodologies to promote comprehensive sequencing of intact multi-protein complexes, 2) leverage next-generation cyclic ion mobility-mass spectrometry (IM-MS) technology to produce high-definition collision induced unfolding (CIU) and native top-down sequencing methods that enable improved identification of proteoforms within assemblies, 3) produce new techniques for the direct sequencing of membrane protein complexes, including laser-based activation of detergent clusters for improved data quality, 4) combine electron capture dissociation (ECD) with CIU for on-the-fly annotation of fingerprint data, and 5) automated methods for protein derivatization and clean-up compatible with native proteomics. This technology will be brought to bear to discover the structures of a series of selected proteoforms and complexes, each linked to human disease.