Practical Mass Spectrometer Upgrade for Identifying Fragile Protein Modifications by ECD

摘要：现代质谱仪的速度、分辨率和质量精度都发生了革命性的变化 蛋白质组学，但准确识别和量化翻译后修饰(PTM) 仍然是一个重大挑战，最终限制了许多当前的生物医学和制药应用。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 碎裂，因为大多数PTM，如磷酸化，具有不稳定的键，通常在 当受到CID时，方式复杂。此外，CID将蛋白质组学限制在对胰酶的自下而上的分析- 10-40个残基的消化多肽。众所周知，另一种碎片化方法 所谓的电子俘获解离(ECD)可以产生特别干净的光谱，以保存PTM，但 这项技术目前只在昂贵的FTICR质谱仪上可行。提供足够低的- 到目前为止，能量电子有效地碎裂多肽的应用从根本上限制了 ECD。我们已经开发出一种ECD电池，它的运行不会影响常规质量的离子飞行路径 分光计。基于这项新技术，我们的第一阶段SBIR项目被设计为至少将 利用Orbitrap质谱仪独特的几何结构实现碎裂效率 离子使其两次通过ECD电池。我们超过了第一阶段的里程碑，证明了 我们的ECD电池的效率翻了两番，部分原因是离子在Orbitrap中通过电池的速度比 在其他类型的质谱计中。我们进一步证明，我们的ECD单元可以很容易地安装在Orbitraps中 在不影响乐器性能的情况下，在一个小时内。我们把ECD的工作建立得特别好 用于天然蛋白质的分析，甚至用于自上而下的氢/氢结构分析。对于第二阶段， 我们的第一个目标是改进ECD单元中的每个元素，以便轻松集成到四个Orbitrap系列中 然后利用细胞产生高能电子的能力来实现更强的 电子诱导解离(EID)碎裂。我们的第二个目标是与早期采用者合作 开发用于商业发布的技术并验证其相对于竞争对手的实质性优势 技术。采用我们的技术将加快许多NIH调查人员调查 疾病机制和识别诊断/治疗生物标记物的速度和准确性都有所提高 将减少复杂生物样本中的错误识别。我们马上要做的广告 第三阶段的目标是为服役中的6000个轨道器提供成本效益高的升级套件。时间越长- Range的商业目标是开发完全集成的解决方案，使生物制药 工业表征治疗性蛋白质产品，如抗体结合药物，并验证 FDA和其他监管机构的“生物仿制药”。

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.