Practical Mass Spectrometer Upgrade for Identifying Fragile Protein Modifications by ECD

用于通过 ECD 识别脆性蛋白质修饰的实用质谱仪升级

• 批准号: 9542850
• 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: 2017-01-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542850
• 关键词: Acetylation; Address; Adoption; Affect; Antibody-drug conjugates; Arthritis; Australia; Biological; Biological Products; Cells; Charge; Chronic Disease; Cleaved cell; Complex; Computer software; Detection; Deuterium; Diabetes Mellitus; Diagnosis; Diagnostic; Disease; Dissociation; Electrons; Elements; Europe; Family member; Feedback; Foundations; Fourier transform ion cyclotron resonance; Geometry; Goals; Heart Diseases; Hour; Hydrogen; Industry; Inflammation; Ions; Isotopes; Laboratories; Learning; Letters; Logistics; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Methodology; Methods; Mission; Modeling; Modernization; Modification; Nerve Degeneration; Pathway interactions; Peptide Fragments; Peptides; Performance; Pharmacologic Substance; Phase; Phosphorylation; Play; Post-Translational Protein Processing; Power Sources; Process; Proteins; Proteomics; Research Personnel; Resolution; Role; Sampling; Services; Shapes; Small Business Innovation Research Grant; Source; Speed; Techniques; Technology; Testing; Therapeutic Intervention; Tissues; Travel; Trypsin; United States; United States National Institutes of Health; Work; base; commercialization; cost effective; density; design; electron energy; experience; improved; insight; instrument; magnetic field; mass spectrometer; new technology; next generation; prospective; prototype; research and development; success; therapeutic biomarker; therapeutic protein; tool

Summary: The speed, resolution, and mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantification of post-translational modifications (PTMs) remain a major challenge that ultimately limits many current biomedical and pharmaceutical applications. A pivotal weakness lies in the almost exclusive use of collision-induced dissociation (CID) to induce fragmentation because most PTMs, such as phosphorylation, have labile bonds that are commonly lost in complex ways when subjected to CID. Furthermore, CID limits proteomics to bottom-up analyses of trypsin- digested peptides of 10-40 residues. It is well established that an alternative fragmentation methodology called electron capture dissociation (ECD) can produce exceptionally clean spectra that preserve PTMs, but this technique is currently feasible only in expensive FTICR mass spectrometers. Providing enough low- energy electrons to efficiently fragment peptides has, until now, fundamentally limited the application of ECD. We have developed an ECD cell that operates without affecting the ion-flight path of conventional mass spectrometers. Based on that new technology, our Phase I SBIR project was designed to at least double fragmentation efficiency by exploiting the distinctive geometry of Orbitrap mass spectrometers to enable ions to make two passes through the ECD cell. We exceeded our Phase I milestones by demonstrating that our ECD cell quadrupled efficiency, due in part to ions moving slower through our cell in the Orbitrap than in other types of mass spectrometers. We further showed that our ECD cell was easily installed in Orbitraps in an hour without affecting the instruments' performance. We established the ECD works particularly well for the analysis of native proteins, even for top-down hydrogen/deuterium structural analyses. For Phase II, our 1st aim is to refine each of the elements in the ECD cell to integrate easily in four Orbitrap family members and then to exploit the cell's capabilities to produce high-energy electrons to achieve stronger fragmentation by Electron-Induced Dissociation (EID). Our 2nd aim involves working with early-adopters to develop the technology for commercial release and validate its substantial advantages over competing technologies. Adoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic/therapeutic biomarkers with increased speed and accuracy that will result in fewer mistaken identifications in complex biological samples. Our immediate commercial objective for Phase-III is to provide cost-effective upgrade kits for the 6,000 Orbitraps in service. The longer- range commercial goal is to develop fully integrated solutions that will enable the biopharmaceutical industry to characterize therapeutic protein products such as antibody-conjugated drugs, and to validate “biosimilars” for the FDA and other regulatory agencies.

摘要：现代质谱仪的速度、分辨率和质量精度发生了革命性的变化 蛋白质组学，但翻译后修饰 (PTM) 的准确识别和定量 仍然是最终限制许多当前生物医学和制药应用的重大挑战。一个 关键的弱点在于几乎完全使用碰撞诱导解离（CID）来诱导 断裂，因为大多数 PTM（例如磷酸化）具有不稳定的键，这些键通常会在 接受 CID 时的复杂方式。此外，CID 将蛋白质组学限制为胰蛋白酶的自下而上分析。 10-40 个残基的消化肽。众所周知，另一种碎片方法 称为电子捕获解离 (ECD) 的技术可以产生异常干净的光谱，从而保留 PTM，但是 该技术目前仅在昂贵的 FTICR 质谱仪中可行。提供足够的低 迄今为止，能量电子有效裂解肽的应用从根本上限制了 ECD。我们开发了一种 ECD 单元，其运行时不会影响常规质量的离子飞行路径 光谱仪。基于这项新技术，我们的第一阶段 SBIR 项目设计为至少增加一倍 通过利用 Orbitrap 质谱仪独特的几何结构来提高碎片效率 离子两次通过 ECD 池。我们通过证明超越了第一阶段的里程碑 我们的 ECD 细胞效率提高了四倍，部分原因是离子在 Orbitrap 中通过细胞的速度比 在其他类型的质谱仪中。我们进一步表明我们的 ECD 单元可以轻松安装在 Orbitraps 中 一个小时内完成，不会影响仪器的性能。我们建立的 ECD 效果特别好 用于分析天然蛋白质，甚至用于自上而下的氢/氘结构分析。对于第二阶段， 我们的第一个目标是完善 ECD 单元中的每个元素，以便轻松集成到四个 Orbitrap 系列中 成员，然后利用细胞产生高能电子的能力来实现更强的 通过电子诱导解离 (EID) 产生碎片。我们的第二个目标是与早期采用者合作 开发商业发布技术并验证其相对于竞争者的显着优势 技术。采用我们的技术将加速许多 NIH 研究人员的探索能力 疾病机制并以更高的速度和准确性识别诊断/治疗生物标志物 将减少复杂生物样品中的错误识别。我们的即时商业 第三阶段的目标是为正在使用的 6,000 个 Orbitrap 提供具有成本效益的升级套件。越长—— 系列商业目标是开发完全集成的解决方案，使生物制药 行业来表征治疗性蛋白质产品，例如抗体偶联药物，并验证 FDA 和其他监管机构的“生物仿制药”。