Hybrid SID, IM, and UVPD Methods for Complex-Down MS of Protein Complexes

用于蛋白质复合物复合降低 MS 的混合 SID、IM 和 UVPD 方法

• 批准号: 10441401
• 负责人: Joshua David Gilbert
• 金额: $ 16.91万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441401
• 关键词: Automobile Driving; Binding; Binding Sites; Biological Process; Cells; Collection; Communities; Complex; Coupled; Coupling; Custom; Development; Devices; Dimensions; Disease; Dissociation; Fourier transform ion cyclotron resonance; Hybrids; Individual; Ions; Laboratories; Ligand Binding; Ligands; Mass Spectrum Analysis; Membrane Proteins; Methods; Minor; Molecular Conformation; Nucleoproteins; Peptides; Performance; Periodicity; Post-Translational Protein Processing; Process; Protein Subunits; Proteins; Research Personnel; Resolution; Resources; Sampling; Shapes; Site; Spectrometry; Speed; Structural Models; Structure; Surface; System; Techniques; Technology; Time; Tube; Vendor; Vertebral column; Water; base; experimental study; improved; instrument; interest; ion mobility; mass analyzer; mobility aid; protein complex; prototype; stoichiometry; structural biology; tool; transmission process; ultraviolet

In order to fulfill their functions many protein complexes must first form dynamic complexes with multiple other proteins or binding partners. Characterization of the overall stoichiometry, topology, and inter- and intra-subunit contacts of protein and nucleoprotein complexes, and their assembly/disassembly, is critical because these complexes regulate key biological processes. Native mass spectrometry (nMS), particularly in combination with ion mobility (IM), and activation methods such as collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and surface induced dissociation (SID), is emerging as a powerful technique with which to study these complex systems and for guiding appropriate application of other structural biology tools. Despite the promise of nMS for structural biology, commercial instruments lack many of the tools necessary to fully characterize these complexes. SID, which is not yet commercialized, has proven to be an incredibly useful tool in the study of protein complexes, cleaving the weakest interfaces in the complex and producing sub- complexes that are reflective of the structure’s connectivity. While IM is commercially available on some platforms, the resolution is often insufficient for detailed structural studies. In TR&D1, we propose to enable higher energy SID to be performed, with more efficient fragment ion collection on multiple different instrument platforms. In TR&D2 we propose to develop high-resolution IM on a high-resolution Orbitrap instrument. In this TR&D we propose to couple the technologies developed in TR&Ds1 and 2, in addition to vendor prototype IM devices, in order to enable the full characterization of protein complexes using integrated, efficient workflows. Coupling of SID and IM is essential because when SID is placed before IM it allows conformational information to be obtained on the intact complex and the subcomplexes produced from SID even when the peaks overlap in m/z space, enabling structural models to be built. When SID is placed after IM, it allows different conformations of the intact complex (if present) to be individually mobility-selected for fragmentation. In addition to coupling SID and IM, we propose to combine these approaches with UVPD. This allows for the interrogation of complex assembly and subunit connectivity with SID and IM, with covalent fragmentation (sequencing of the peptide backbone) from UVPD. This approach will be beneficial in discerning ligand binding sites along with the sites of any post-translational modifications (PTMs). We propose to do this on multiple instrumental platforms, including the Waters Synapt G2(S), Thermo (Q) Exactive, and Bruker FTICR. The use of multiple platforms is necessary as each platform has different mass resolution, sensitivity, and speed, and certain platforms will be better suited to certain complexes. Hence incorporation with multiple platforms allows the experiments to be customized to the complex of interest. The use of multiple platforms is also essential as it allows better dissemination to the wider community, who may have access to only one of these platforms.

为了实现其功能，许多蛋白质复合物必须首先与多种其他蛋白质形成动态复合物 蛋白质或结合伴侣的总体化学计量、拓扑结构以及亚基间和亚基内的表征。 蛋白质和核蛋白复合物的接触及其组装/拆卸至关重要，因为这些 复合物调节关键的生物过程，特别是与天然质谱（nMS）结合。 离子淌度 (IM) 和碰撞诱导解离 (CID)、紫外线等激活方法 光解离 (UVPD) 和表面诱导解离 (SID) 正在成为一种强大的技术， 研究这些复杂的系统并指导其他结构生物学工具的适当应用。 尽管 nMS 在结构生物学方面前景广阔，但商业仪器缺乏许多必要的工具 充分表征这些复合物，虽然尚未商业化，但已被证明是非常有用的。 研究蛋白质复合物的工具，切割复合物中最弱的界面并产生子 反映结构连通性的复合体，而 IM 在某些产品上已上市。 平台上，分辨率通常不足以进行详细的结构研究，我们建议启用。 执行更高能量的 SID，在多个不同仪器上进行更有效的碎片离子收集 在 TR&D2 中，我们建议在高分辨率 Orbitrap 仪器上开发高分辨率 IM。 TR&D 除了供应商原型 IM 之外，我们建议结合 TR&D1 和 2 中开发的技术 设备，以便使用集成、高效的工作流程来全面表征蛋白质复合物。 SID 和 IM 的耦合至关重要，因为当 SID 置于 IM 之前时，它允许构象信息 即使峰重叠，也可以在完整复合物和 SID 产生的子复合物上获得 m/z 空间，可以构建结构模型。当 SID 放置在 IM 之后时，它允许不同的构象。 除了偶联 SID 之外，还可以对完整复合物（如果存在）进行单独的迁移率选择。 和 IM，我们建议将这些方法与 UVPD 结合起来，这样可以探究复杂性。 与 SID 和 IM 的组装和亚基连接，以及共价断裂（肽测序） 来自 UVPD 的主链）这种方法将有利于识别配体结合位点以及配体结合位点。 我们建议在多个工具平台上进行此操作，包括 Waters Synapt G2(S)、Thermo (Q) Exactive 和 Bruker FTICR 需要使用多个平台。 由于每个平台具有不同的质量分辨率、灵敏度和速度，并且某些平台会更适合 因此，与多个平台的结合允许定制实验。 兴趣综合体的使用也很重要，因为它可以更好地传播给大众。 更广泛的社区，他们可能只能访问这些平台之一。