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

近年来，由于技术的进步，本地质谱分析的能力得到了显着提高 仪器速度、分辨率、质量精度和专门设计用于处理大蛋白质的离子光学器件 配合物尽管有这些进步，但通过天然质谱法可实现的表征深度仍然是有限的。 由于质量内的大蛋白质复合物的低效解离和片段化， 光谱仪在e-MSion公司，我们开发了一种高效的电子分裂技术，称为ExD 现在与Agilent共同销售其Q-TOF系列，与沃茨共同销售其Q-IM-TOF，与 他们的超高质量范围（UHMR）轨道器。ExD技术提供广泛的 变性和天然蛋白质的片段化，使得能够彻底测序和定位 翻译后修饰然而，许多蛋白质复合物的大质量，现在可以通过 质谱分析使得它们特别难以被基于电子离子解离和碎片化 单独激活方法。表面诱导解离（SID）是一种能够 解离大的蛋白质复合物以揭示更高级的结构，例如亚基化学计量， 拓扑和接口，具有最小的子单元的展开和碎片化。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 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。 俄亥俄州州立大学的威索基实验室的最新进展导致了 他们的SID设计，这使得联合收割机结合两种互补的方法， 蛋白质解离和片段化。在第一阶段的建议中，我们将评估开发 用于UHMR Orbitrap质谱仪的混合ExD-SID池，用于表征二硫化物交联的天然 蛋白质复合物执行SID所需的离子光学器件和电子器件将集成到ExD单元中 UHMR Orbitrap平台的ExD控制器。我们将优化混合电池设计， 传输、ExD、SID和ExD-SID实验。所开发的混合电池和方法将被应用于 将天然抗体表征为模型系统。混合ExD-SID小区将实现高效的 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一个 是快速表征和发现单克隆抗体治疗剂的有力工具。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 光谱法进入主流的结构生物学方法，大大扩大了质量范围， 大分子复合物适合广泛的表征。

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.