Enabling electron-induced fragmentation in tandem mass spectrometry

摘要：现代质谱仪的速度、分辨率和高质量准确度， 彻底改变了蛋白质组学，但准确识别和定量的翻译后 修饰（PTM）仍然是一个主要的挑战--对于许多重要的医学应用来说是一个关键的限制。 目前用于蛋白质组学的质谱法的一个关键弱点在于用于诱导蛋白质组学的方法。 片段化，因为磷酸化等PTM是蛋白质中最不稳定的化学键之一 并且通过当前基于碰撞的碎片化方法以复杂的方式丢失。一个替代 被称为电子捕获解离（ECD）的碎裂方法被很好地建立以产生 保留PTM的异常干净的光谱，但目前仅在昂贵的FTICR质量中可行 光谱仪ECD的基本限制是难以提供足够的低能电子 以有效地将肽片段化。我们已经发现了如何使用精心塑造的磁场与热 产生电子的细丝，以抑制离子飞行路径中的大量电子。这可以 适用于任何常见的串联质谱仪，而不改变现有的离子光学，但我们最好的 设计只能片段化3-5%的双电荷胰蛋白酶消化的肽-最常见的工作流程 用于质谱分析。这种低碎裂效率限制了灵敏度，这已被证明是 这是质谱行业采用这种强大方法的主要障碍。的主要重点 这个SBIR项目的第一阶段是确定如何增加离子与受限电子的相互作用时间 通过磁场将光束转换为窄光束来证明这一概念的可行性。目前的反应时间是1-2 微秒。我们的第一阶段可行性问题是，碎片化是否可以有效地增加， 通过在ECD池中瞬时停止肽离子而没有由于静电引起的显著损失， 散射此外，设计必须保持亚毫秒的速度，以兼容 目前用于复杂样品的质谱仪的前端HPLC和离子迁移率分离。 严格的计算机模拟表明，这些目标可以通过仔细冷却前体来实现。 离子，然后通过仔细定时的电脉冲暂时停止它们的飞行到静电透镜。 可行性证明和经过验证的概念演示（第二阶段）对于吸引主要 仪器制造商进一步开发和商业化我们的ECD技术，用于他们的大规模 光谱仪产品。成功还将展示我们的技术如何能够更好地分解 质谱分析最具挑战性的分析物，包括脂质、聚糖和其他难以分析的 碎片药物/代谢物。采用我们的技术将加速许多NIH 研究人员探索疾病机制，并确定诊断/治疗生物标志物， 准确性和更高的速度，同时在复杂的生物样品中进行更少的错误鉴定。

Summary: The speed, resolution and high mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantitation of post-translational modifications (PTMs) remain a major challenge—a key limitation for many important medical applications. A key weakness with current mass spectrometry for proteomics lies in the methods used to induce fragmentation, because PTMs such as phosphorylation are among the most labile chemical bonds in proteins and are lost in complex ways by current collision-based fragmentation approaches. An alternative fragmentation methodology called electron capture dissociation (ECD) is well established to produce exceptionally clean spectra that preserve PTMs, but is currently feasible only in expensive FTICR mass spectrometers. The fundamental limitation to ECD is the difficulty of providing enough low-energy electrons to efficiently fragment peptides. We have discovered how to use carefully sculpted magnetic fields with a hot electron-producing filament to restrain large numbers of electrons in the flight path of ions. This can be adapted in any common tandem mass spectrometer without changing the existing ion optics, but our best designs can only fragment 3-5% of doubly charged trypsin-digested peptides—the most common workflow used in mass spectrometry. This low fragmentation efficiency limits sensitivity, which has proved to be the major barrier to adopting this powerful methodology by the mass spectrometry industry. The key focus of this Phase I SBIR project is determining how to increase the interaction time of ions with electrons confined to a narrow beam by the magnetic fields to prove this concept feasible. The reaction time currently is 1-2 microseconds. Our Phase I feasibility question is whether fragmentation can be effectively increased at least two-fold by transiently stopping peptide ions in the ECD cell without significant loss due to electrostatic scattering. In addition, the design must retain the sub-millisecond speed necessary to be compatible for current front-end HPLC and ion mobility separations used with mass spectrometers for complex samples. Rigorous computer simulations show these objectives can be accomplished by carefully cooling precursor ions and then transiently stopping their flight with carefully timed electrical pulses to electrostatic lenses. Proof of feasibility and validated concept demonstration (Phase II) are essential in engaging the major instrument manufacturers to further develop and commercialize our ECD technology for use in their mass spectrometer products. Success will also show how our technology can produce better fragmentation of the most challenging analytes analyzed by mass spectrometry, including lipids, glycans, and other difficult-to- fragment drugs/metabolites. The adoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic/therapeutic biomarkers with increased accuracy and greater speed, while making fewer mistaken identifications in complex biological samples.