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.

天然质谱学的能力在最近几年有了显著的提高，这是由于在 仪器的速度、分辨率、质量精度和专为处理大蛋白质而设计的离子光学 复合体。尽管有这些进展，自然质谱学可以实现的表征深度是 由于团块内大型蛋白质复合体的低效解离和碎裂，仍然受到限制 分光计。在e-MSion，Inc.，我们开发了一种名为EXD的高效电子碎片技术 现在与安捷伦共同营销其Q-TOF系列，与Waters共同营销其Q-IM-TOF，以及与 他们的超高质量范围(UHMR)轨道陷阱的热。EXD技术提供了广泛的 变性蛋白和天然蛋白的裂解使其能够进行彻底的测序和定位 翻译后修饰。然而，许多蛋白质复合体的大量现在可以通过 质谱学使它们特别难被电子基离子解离和碎裂 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体以揭示更高级的结构，如亚单位化学计量比， 拓扑和接口，亚基的展开和碎片化最小。然而，SID是 不能分离分子间二硫键交联蛋白。我们已经展示了我们的EXD技术是 在切割半胱氨酸结蛋白和单抗中的多个二硫键方面非常有效。 俄亥俄州立大学Wysocki实验室的最新进展导致显著缩短了 它们的SID设计，现在可以将本机的两种互补方法结合在一起 蛋白质解离和碎裂。在这一阶段的提案中，我们将评估发展的可行性 用于UHMR Orbitrap质谱仪的混合EXD-SID池用于表征二硫键交联天然产物 蛋白质复合体。执行SID所需的离子光学和电子学将集成到EXD单元中 以及用于UHMR Orbitrap平台的EXD控制器。我们将优化混合电池的设计，以最大限度地提高离子 传输、EXD、SID和EXD-SID实验。所开发的混合电池和方法将被应用 将天然抗体作为模型系统进行表征。混合EXD-SID电池将实现高效 二硫键的断裂和非共价相互作用的解离使得能够分离 完整的抗体重链和轻链。成功地解决可行性问题将产生一个 快速鉴定和发现单抗疗法的有力工具。更广泛地说， 混合EXD-SID电池的成功开发将创造一种能够带来原生质量的工具 通过极大地扩展光谱的质量范围，使其成为结构生物学方法的主流 可进行广泛表征的大分子络合物。