Advancing native top-down protein analysis with hybrid SID/ECD technology

利用混合 SID/ECD 技术推进天然自上而下蛋白质分析

• 批准号: 10155281
• 负责人: Jared Bryan Shaw
• 金额: $ 25.21万
• 依托单位: E-MSION, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10155281
• 关键词: Address; Antibodies; Arthritis; Biological Models; Biological Products; Biological Response Modifier Therapy; Businesses; Capsid; Cell surface; Cells; Complex; Cysteine; Development; Devices; Disease; Dissociation; Disulfides; Electronics; Electrons; Family; Higher Order Chromatin Structure; Hour; Hybrid Cells; Hybrids; Individual; Ions; Laboratories; Left; Light; Macromolecular Complexes; Mainstreaming; Marriage; Mass Spectrum Analysis; Methods; Monoclonal Antibodies; Nature; Nerve Degeneration; Ohio; Optics; Performance; Periodicity; Phase; Positioning Attribute; Post-Translational Protein Processing; Protein Analysis; Proteins; Proteomics; Reaction; Reporting; Resolution; Small Business Innovation Research Grant; Speed; Sulfhydryl Compounds; Surface; Techniques; Technology; Therapeutic Monoclonal Antibodies; Therapeutic antibodies; Thinness; Universities; Viral; Viral Respiratory Tract Infection; Water; Work; base; cost effective; crosslink; design; design and construction; detection sensitivity; disulfide bond; experimental study; flexibility; frontier; improved; instrument; lens; link protein; mass spectrometer; miniaturize; novel; preservation; protein complex; protein crosslink; stoichiometry; structural biology; success; tool; transmission process

The capabilities of native mass spectrometry have improved dramatically in recent years due to advances in instrument speed, resolution, mass accuracy, and ion optics designed specifically to handle large protein complexes. Despite these advances, the depth of characterization achievable by native mass spectrometry is still limited due to inefficient dissociation and fragmentation of large protein complexes within the mass spectrometer. At e-MSion, Inc., we have developed an efficient electron-fragmentation technology called ExD now co-marketed with Agilent for their family of Q-TOFs, with Waters for their Q-IM-TOFs, and with Thermo for their Ultra High Mass Range (UHMR) Orbitraps. The ExD technology provides extensive fragmentation of denatured and native proteins enabling thorough sequencing and localization of posttranslational modifications. However, the large masses of many protein complexes now accessible by mass spectrometry make them particularly challenging to dissociate and fragment by electron-based ion activation methods alone. Surface induced dissociation (SID) is a complementary technique capable of dissociating large protein complexes to reveal higher order structure, such as subunit stoichiometry, topology, and interfaces, with minimal unfolding and fragmentation of the subunits. However, SID is incapable of separating intermolecular disulfide crosslinked proteins. We have shown our ExD technology is extremely effective at cutting multiple disulfide bonds in Cysteine Knot Proteins and monoclonal antibodies. Recent advances in the Wysocki lab at Ohio State University have resulted in a remarkable shortening of their SID design, which now makes it possible to combine the two complementary approaches of native protein dissociation and fragmentation. In this phase I proposal, we will evaluate the feasibility of developing a hybrid ExD-SID cell for the UHMR Orbitrap mass spectrometer to characterize disulfide-crosslinked native protein complexes. Ion optics and electronics required to perform SID will be integrated into the ExD cell and ExD controller for the UHMR Orbitrap platform. We will optimize the hybrid cell design to maximize ion transmission, ExD, SID, and ExD-SID experiments. The developed hybrid cell and methods will be applied the characterization of native antibodies as a model system. The hybrid ExD-SID cell will enable efficient fragmentation of disulfide bonds and dissociation of noncovalent interactions enabling separation of the intact heavy and light chains of the antibody. Success in addressing the feasibility question will yield a powerful tool for rapid characterization and discovery of monoclonal antibody therapeutics. More broadly, successful development of a hybrid ExD-SID cell will create a tool capable of bringing native mass spectrometry into the mainstream for structural biology approaches by greatly expanding the mass range of macromolecular complexes amenable to extensive characterization.

近年来，由于技术的进步，天然质谱分析的能力得到了显着提高。 专为处理大蛋白质而设计的仪器速度、分辨率、质量精度和离子光学器件 复合物。尽管取得了这些进步，但通过原生质谱法可实现的表征深度仍然有限​​。 由于质量内大型蛋白质复合物的低效解离和断裂，仍然受到限制 光谱仪。在 e-MSion, Inc.，我们开发了一种称为 ExD 的高效电子碎片技术 现在与 Agilent 共同销售其 Q-TOF 系列，与 Waters 共同销售其 Q-IM-TOF，并与 Thermo 的超高质量范围 (UHMR) Orbitraps。 ExD 技术提供了广泛的 变性和天然蛋白质的片段化能够实现彻底的测序和定位 翻译后修饰。然而，现在可以通过以下方式获得大量的许多蛋白质复合物： 质谱分析使它们特别难以通过电子离子解离和碎片 单独的激活方法。表面诱导解离 (SID) 是一种补充技术，能够 解离大的蛋白质复合物以揭示更高阶的结构，例如亚基化学计量， 拓扑和界面，子单元的展开和碎片最小化。然而，SID 是 不能分离分子间二硫键交联的蛋白质。我们已经证明我们的 ExD 技术是 在切割半胱氨酸结蛋白和单克隆抗体中的多个二硫键方面非常有效。 俄亥俄州立大学 Wysocki 实验室的最新进展显着缩短了 他们的 SID 设计，现在可以结合本机的两种互补方法 蛋白质解离和断裂。在第一阶段提案中，我们将评估开发的可行性 用于 UHMR Orbitrap 质谱仪的混合 ExD-SID 细胞，用于表征二硫键交联的天然 蛋白质复合物。执行 SID 所需的离子光学和电子器件将集成到 ExD 单元中 和 UHMR Orbitrap 平台的 ExD 控制器。我们将优化混合电池设计以最大化离子 传输、ExD、SID 和 ExD-SID 实验。开发的混合细胞和方法将被应用 作为模型系统的天然抗体的表征。混合 ExD-SID 单元将实现高效 二硫键的断裂和非共价相互作用的解离使得能够分离 抗体的完整重链和轻链。成功解决可行性问题将产生 用于快速表征和发现单克隆抗体疗法的强大工具。更广泛地说， 混合 ExD-SID 细胞的成功开发将创造一种能够带来天然质量的工具 光谱分析法通过极大地扩展质量范围而成为结构生物学方法的主流 适合广泛表征的大分子复合物。