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和 他们在生物制药行业的翻译后修饰（PTM）。这些分析仍 尽管现代质量的速度、分辨率和质量精度取得了重大进步，但仍具有挑战性 光谱仪。当前蛋白质表征仪器的一个关键弱点在于方法 用于诱导碎片。特别是对碰撞诱导解离 (CID) 的依赖受到了限制 此类分析适用于胰蛋白酶消化的 10-30 个残基肽的自下而上工作流程。当受到 CID，这些短肽上的许多脆弱的 PTM 以复杂的方式丢失。另一种碎片化方式 电子捕获解离 (ECD) 方法因生产极其清洁的产品而闻名 整个蛋白质的光谱，同时还保留 PTM。然而，这项技术仅在 昂贵的 FTICR 质谱仪。困难在于限制足够的低能电子 有效地断裂肽键，这限制了 ECD 在其他仪器中的应用。这 e-MSion 团队开发了一种高效的 ECD 技术，通过精心设计的结构来限制电子 不影响质谱仪中离子飞行路径的磁场。一大优势 与 ETD 等竞争性碎片技术相比，我们技术的优势在于速度。我们建立了一期 通过证明我们的 ECD 技术足够快，可以用于四极飞行时间 (Q- ToF）质谱仪，速度与 UPLC 和基于离子淌度的复杂化合物分离兼容 样品。我们的技术还有效支持在几秒钟内对大至 30 kDa 的蛋白质进行测序，同时 即使是最脆弱的 PTM 也能完好无损。拟议的第二阶段 SBIR 项目将完成 将我们的 ECD 优化/集成到 Q-ToF 中，使两个主要的操作无缝进行 Q-ToF 制造商。主要商业目标是成为升级Q-的增值经销商 Tofs 处于第三阶段。为了实现这一目标，我们的首要目标是完善工程、软件集成和 应用到中层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 生物制药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 当前的 ECD 电池允许大量高能电子流过系统。 Aim 3 的完成将打开三重四极杆质谱仪的市场，其规模是其五倍 Q-ToF 更昂贵。采用我们的技术将加速许多 NIH 的能力 研究人员和生物制药公司通过表征大分子来探索疾病机制 提高复杂生物样本的准确性和速度，同时减少错误发现。