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

项目摘要 在每个生物体中，蛋白质都在发挥作用，进行影响细胞功能各个方面的活动。 实现如此广泛的蛋白质功能的关键因素涉及翻译后修饰 和序列修饰，以产生大量的功能蛋白质状态，形成一个单一的基因， 产品这种蛋白形式进一步直接偶联到所产生的生物分子的3D结构， 被进一步招募到各种各样的动态多蛋白质机器中。直接评估的结构 这些组装状态，沿着它们所包含的蛋白形式，对于理解人类 疾病尽管如此，大多数结构仍然是未知的，并且对当前技术是难熔的，并且它们的 蛋白形式的互补物保持不透明。标准结构生物学方法（X射线、NMR和Cryo-EM）， 虽然非常成功，但需要大量的纯样品，精心优化以生产 单分散性蛋白质群体的各个方面，并消除光谱背景。此外，瞬态 并且存在于复杂混合物中的多分散组件不能被分析。质谱法（MS） 为解决这一具有挑战性的问题而开发的方法可以克服这些障碍中的许多。虽然这些 工具正在经历一个快速的发展阶段，他们目前缺乏谨慎评估的能力， 蛋白质型对多蛋白质组构的影响。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， 组成和三维结构的主体的动态，异质大分子复合物的明确 生物医学的重要性 该更新申请寻求构建新的、创新的MS技术，其1）利用新的类 化学标记试剂和混合标记方法，以促进完整的 多蛋白质复合物，2）利用下一代循环离子迁移率质谱（IM-MS） 产生高清晰度碰撞诱导解折叠（CIU）和天然自上而下测序的技术 能够改进组件内蛋白质型鉴定的方法，3）产生新技术， 膜蛋白复合物的直接测序，包括基于激光的去污剂簇的活化 为了提高数据质量，4）将联合收割机电子捕获解离（ECD）与CIU结合，用于实时注释 指纹数据，和5）用于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来发现一系列选定的结构 蛋白形式和复合物，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.