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，分辨率和质量准确性已革新 蛋白质组学，但经过翻译后修饰（PTM）的准确识别和量化 仍然是一个主要挑战，最终限制了许多当前的生物医学和药物应用。一个 关键弱点在于几乎独有地使用碰撞诱导的解离（CID）来诱导 分散化，因为大多数PTM（例如磷酸化）具有不稳定的键，这些键通常在 遭受CID的复杂方式。此外，CID将蛋白质组学限制为对胰蛋白酶的自下而上分析 10-40个残留物的消化肽。众所周知，一种替代性碎片方法 称为电子捕获解离（ECD）可以产生特殊的清洁光谱来保留PTM，但 目前，此技术仅在昂贵的FTICR质谱仪中可行。提供足够的低 - 到目前为止 ECD。我们已经开发了一个ECD单元，该电池可在不影响常规质量的离子飞行路径的情况下运行 光谱仪。基于这项新技术，我们的I阶段SBIR项目旨在至少两倍 通过利用Orbitrap质谱仪的独特几何形状来实现碎片化效率 离子可以通过ECD细胞进行两个通过。我们通过证明这一点超出了I阶段里程碑 我们的ECD细胞四倍体效率，部分是由于离子在Orbitrap中的细胞慢移动而不是 在其他类型的质谱仪中。我们进一步表明，我们的ECD单元很容易安装在Orbitraps中 在一个小时内，不会影响乐器的性能。我们确定了ECD的运作良好 对于第二阶段， 我们的第一个目标是完善ECD单元格中的每个元素，以轻松整合四个Orbitrap家族 成员，然后利用细胞产生高能电子的能力以实现强大 电子诱导的解离（EID）的碎片化。我们的第二个目标涉及与早期管理员合作 开发商业发布的技术，并验证其与竞争的实质优势 技术。采用我们的技术将加速许多NIH调查人员证明的能力 疾病机制并以提高速度和准确性鉴定诊断/治疗生物标志物 在复杂的生物样品中的错误识别将减少。我们的直接广告 III期的目的是为6,000个服务中的Orbitrap提供具有成本效益的升级套件。更长 范围的商业目标是开发完全集成的解决方案，以实现生物制药 旨在表征治疗性蛋白质产品（例如抗体偶联药物）的行业，并验证 FDA和其他监管机构的“生物仿制药”。