Enabling electron-induced fragmentation in tandem mass spectrometry

在串联质谱分析中实现电子诱导碎裂

• 批准号: 9751318
• 负责人: Valery G. Voinov
• 金额: $ 74.63万
• 依托单位: E-MSION, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751318
• 关键词: Address; Adoption; Affect; Arthritis; Biological; Biological Products; Biological Response Modifier Therapy; Cardiovascular Diseases; Cells; Charge; Chimeric Proteins; Cleaved cell; Complex; Computer software; Computers; Consumption; Deuterium; Development; Diabetes Mellitus; Diagnosis; Disease; Dissociation; Electron Transport; Electrons; Engineering; Fourier transform ion cyclotron resonance; Glycopeptides; Glycoproteins; Goals; Heart Diseases; Hour; Human; Hydrogen; Industry; Inflammation; Interview; Ions; Isotopes; Legal patent; Malignant Neoplasms; Manufacturer Name; Marketing; Mass Spectrum Analysis; Methodology; Methods; Modernization; Modification; Nerve Degeneration; Pattern; Peptide Fragments; Peptide Sequence Determination; Peptides; Pharmacologic Substance; Phase; Phosphorylation; Polysaccharides; Post-Translational Protein Processing; Production; Protein Fragment; Proteins; Proteomics; Quality Control; Reaction; Recombinant Antibody; Research; Research Personnel; Resolution; Sales; Sampling; Side; Small Business Innovation Research Grant; Speed; System; Techniques; Technology; Time; Tissues; Trypsin; United States National Institutes of Health; Work; base; commercialization; cost; design; electron energy; fly; improved; instrument; instrumentation; ion mobility; ionization; macromolecule; magnetic field; mass spectrometer; next generation; off-patent; operation; phase 1 study; preservation; rapid technique; research and development; success; tandem mass spectrometry; tool

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.

高端质谱仪行业的主要市场焦点是全面表征蛋白质， 翻译后修饰（PTMs）在生物制药行业中的应用。这些分析仍然 尽管现代质量分析在速度、分辨率和质量精度方面取得了重大进展， 光谱仪目前用于蛋白质表征的仪器的关键弱点在于方法 用于诱导分裂。特别是对碰撞诱导解离（CID）的依赖限制了 这种分析是10-30个残基的胰蛋白酶消化肽的自下而上的工作流程。当经受 CID，这些短肽上的许多脆弱的PTM以复杂的方式丢失。另一种分裂 一种被称为电子捕获解离（ECD）的方法， 整个蛋白质的光谱，同时也保留PTM。然而，这项技术只有在 昂贵的FTICR质谱仪。困难来自于限制足够的低能电子， 这限制了ECD在其他仪器中的应用。的 e-MSion团队开发了一种有效的ECD技术，通过精心设计的 在质谱仪中不影响离子飞行路径的磁场。一个主要优点 我们的技术相对于竞争对手的碎片技术（如ETD）的优势在于速度。我们建立了第一阶段 通过显示我们的ECD技术足够快，可以用于四极杆飞行时间（Q- ToF）质谱仪，其速度与UPLC和基于离子迁移率的复合物分离兼容 样品我们的技术还有效地支持在几秒钟内对高达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阶段SBIR项目将完成 优化/整合我们的ECD到Q-ToF的，使两个主要的操作无缝 Q-ToF制造商主要的商业目标是成为一个增值服务商，为升级Q- 第三阶段的Tofs。为了实现这一目标，我们的第一个目标是完善工程、软件集成和 应用于中间和自上而下的蛋白质表征。目标2是与早期采用者合作， Bioburma和蛋白质组学领域展示了该技术的能力。第三个目标是 进一步修改ECD单元的设计以更有效地执行电子诱导解离（EID 用于表征单电荷肽和糖蛋白。这需要对 目前的ECD电池，允许更大量的更高能量的电子流过系统。 目标3的完成将打开三重四重质谱仪的市场，这是五倍于 更昂贵的Q-ToF。采用我们的技术将加速许多NIH 研究人员以及生物制药公司通过表征大分子来探测疾病机制， 复杂的生物样本，提高准确性和速度，同时减少错误的发现。