Enabling electron-induced fragmentation in tandem mass spectrometry

高端质谱仪行业的主要市场焦点是充分表征蛋白质和 他们在生物制药行业内的翻译后修饰(PTM)。这些分析仍然存在 尽管在现代质量的速度、分辨率和质量精度方面取得了重大进展，但仍具有挑战性 分光计。目前用于蛋白质表征的仪器的一个关键弱点在于方法 用来诱导碎裂。尤其对碰撞诱导解离(CID)的依赖是有限的 对胰酶消化的10-30个残基的多肽进行自下而上的工作流程分析。当受制于 CID，这些短肽上的许多脆性PTM以复杂的方式丢失。另一种分割方式 被称为电子俘获解离(ECD)的方法是众所周知的，因为它可以产生异常清洁的 整个蛋白质的光谱，同时也保存了PTM。然而，这项技术只有在 昂贵的FTICR质谱仪。困难在于将足够多的低能电子限制在 这限制了电子捕获技术在其他仪器中的应用。这个 E-MSion团队已经开发出一种高效的ECD技术，通过精心设计的 在质谱仪中运行而不影响离子飞行路径的磁场。一个主要优势 我们的技术相对于竞争对手的碎片化技术(如ETD)的优势在于速度。我们建立了第一阶段 可行性表明，我们的ECD技术足够快，可以用于四极飞行时间(Q-TIME)。 TOF)质谱仪，速度与UPLC和基于离子迁移率的络合物分离兼容 样本。我们的技术还有效地支持在几秒钟内对大至30 kDa的蛋白质进行测序 即使是最脆弱的PTM也完好无损。拟议的第二期SBIR项目将完成 将我们的ECD优化/集成到Q-ToF中，以实现两大业务的无缝运营 Q-TOF的制造商。主要的商业目标是成为Q-升级的增值经销商 第三阶段的TOFS。为了实现这一点，我们的第一个目标是细化工程、软件集成和 应用于中间和自上而下的蛋白质表征。目标2是与这两个领域的早期采用者合作 在生物制药和蛋白质组学领域展示这项技术的能力。第三个目标是 进一步修改ECD电池的设计以更有效地执行电子诱导解离(EID) 用于单电荷肽和糖蛋白的表征。这需要对 当前的ECD电池，允许更大量的高能电子流过系统。 AIM 3的建成将打开三重四级质谱仪的市场，该质谱仪的体积是 价格更高的Q-TOFS。我们技术的采用将加速许多NIH的能力 研究人员以及BioPharma通过表征大分子来探索疾病机制 以更高的准确性和速度处理复杂的生物样本，同时减少错误发现。

The primary market focus for the high-end mass spectrometer industry is to fully characterize proteins and their post-translational modifications (PTMs) within the biopharmaceutical industry. These analyses remain challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation for protein characterization lies in the methods used to induce fragmentation. The reliance in particular on collision-induced dissociation (CID) has limited such analyses to bottom-up workflows of trypsin-digested peptides of 10-30 residues. When subjected to CID, many fragile PTMs on these short peptides are lost in complex ways. An alternative fragmentation methodology called electron capture dissociation (ECD) is well known for producing exceptionally clean spectra of entire proteins while also preserving PTMs. However, this technology has been feasible only in expensive FTICR mass spectrometers. The difficulty arises from confining enough low-energy electrons to efficiently fragment peptide bonds, which has limited the application of ECD in other instruments. The e-MSion team has developed an efficient ECD technology to confine electrons with a carefully designed magnetic field that operates without affecting the ion flightpath in mass spectrometers. One major advantage of our technology over competing fragmentation techniques such as ETD is speed. We established Phase I feasibility by showing that our ECD technology is fast enough to be used in quadrupole-Time of Flight (Q- ToF) mass spectrometers at speeds compatible with UPLC and ion mobility-based separations of complex samples. Our technology also efficiently supports sequencing of proteins as large as 30 kDa in seconds while leaving even the most fragile PTMs intact. The proposed Phase II SBIR project will complete the optimization/integration of our ECD into Q-ToF's to make the operation seamless for two major manufacturers of Q-ToFs. The primary commercial goal is to become a value-added reseller for upgrading Q- Tofs in Phase III. To accomplish this, our first Aim is to refine the engineering, software integration and application to middle- and top-down protein characterization. Aim 2 is to work with early adopters in both Biopharma and in proteomics fields to demonstrate the capabilities of the technology. The third Aim is to further modify the design of the ECD cell to perform Electron-Induced Dissociation (EID) more efficiently for the characterization of singly charged peptides and glycoproteins. This entails subtle modifications to the current ECD cell that allows larger quantities of higher-energy electrons to flow through the system. Completion of Aim 3 will open the market for triple-quad mass spectrometers, which is five times larger than the more expensive Q-ToFs. The adoption of our technology will accelerate the ability of many NIH investigators as well as BioPharma to probe disease mechanisms by characterizing macromolecules in complex biological samples with increased accuracy and speed, while reducing false discoveries.