Enabling electron-induced fragmentation in tandem mass spectrometry

摘要：现代质谱仪的速度、分辨率和高质量精度具有 革命性的蛋白质组学，但准确识别和定量翻译后 修饰(PTM)仍然是一个重大挑战--这是许多重要医学应用的关键限制。 目前蛋白质组学的质谱学的一个关键弱点是用来诱导 碎裂，因为磷酸化等PTM是蛋白质中最不稳定的化学键之一 并且通过当前基于冲突的分段方法以复杂的方式丢失。另一种选择 被称为电子俘获解离(ECD)的碎裂方法被很好地建立起来以产生 特别干净的光谱，保存了PTM，但目前仅在昂贵的FTICR质量中可行 分光计。ECD的根本限制是很难提供足够的低能电子 以有效地碎裂多肽。我们已经发现了如何使用精心雕刻的磁场和热的 产生电子的灯丝，用于抑制离子飞行路径中的大量电子。这可以是 在不改变现有离子光学的情况下适用于任何普通串联质谱仪，但我们最好的 设计只能分割3-5%的双电荷胰酶消化肽-最常见的工作流程 用于质谱学。这种低的碎片效率限制了敏感度，这已被证明是 质谱学行业采用这一强大方法的主要障碍。的主要焦点 这个第一阶段的SBIR项目正在确定如何增加离子与受限电子的相互作用时间 通过对窄束磁场的分析，证明这一概念是可行的。目前的反应时间是1比2 微秒。我们的第一阶段可行性问题是，是否至少可以有效地增加碎片化 通过暂时阻止ECD细胞中的多肽离子而使其加倍，而不会因静电造成显著损失 散布。此外，该设计还必须保持兼容所需的亚毫秒级速度 目前用于复杂样品的前端高效液相色谱和离子迁移率分离与质谱仪一起使用。 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，然后通过仔细计时的电脉冲向静电透镜短暂停止飞行。 可行性证明和经过验证的概念演示(第二阶段)对于与主要 仪器制造商进一步开发和商业化我们的ECD技术，以便在他们的批量使用 光谱仪产品。成功还将展示我们的技术如何更好地产生 通过质谱学分析的最具挑战性的分析物，包括脂类、多糖和其他难以... 碎片药物/代谢物。我们技术的采用将加速许多NIH的能力 研究人员探索疾病机制并确定诊断/治疗生物标记物 精确度和速度更快，同时在复杂的生物样本中错误识别更少。

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.