Enabling electron-induced fragmentation in tandem mass spectrometry

在串联质谱分析中实现电子诱导碎裂

• 批准号: 9346138
• 负责人: Valery G. Voinov
• 金额: $ 22.5万
• 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: 2017-09-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9346138
• 关键词: Acetylation; Address; Adopted; Adoption; Affect; Amides; Biological; Carbohydrates; Cardiovascular Diseases; Cells; Charge; Cleaved cell; Complex; Computer Simulation; Detection; Deuterium; Diagnostic; Disease; Dissociation; Electrons; Electrostatics; Filament; Fourier transform ion cyclotron resonance; Goals; High Pressure Liquid Chromatography; Hydrogen; Industry; Inflammation; Ions; Isotope Labeling; Isotopes; Lipids; Malignant Neoplasms; Manufacturer Name; Mass Spectrum Analysis; Measures; Medical; Methodology; Methods; Modernization; Modification; Molecular; Movement; Nerve Degeneration; Optics; Pattern; Peptide Fragments; Peptides; Pharmaceutical Preparations; Phase; Phosphopeptides; Phosphorylation; Physiologic pulse; Polysaccharides; Post-Translational Protein Processing; Power Sources; Process; Promega; Proteins; Proteomics; Radiation; Reaction Time; Research; Research Personnel; Resolution; Sampling; Small Business Innovation Research Grant; Speed; TRAP Peptide; Technology; Therapeutic Intervention; Time; Tissues; Trypsin; United States National Institutes of Health; Work; base; chemical bond; density; design; disease diagnosis; electron energy; experimental study; improved; instrument; ion mobility; lens; magnetic field; mass spectrometer; millisecond; next generation; programs; research and development; success; tandem mass spectrometry; therapeutic biomarker; tool; transmission process

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.

摘要：现代质谱仪的速度、分辨率和高质量精度已经 彻底改变了蛋白质组学，但翻译后蛋白质的准确识别和定量 修饰（PTM）仍然是一个重大挑战——这是许多重要医疗应用的关键限制。 当前蛋白质组学质谱分析的一个关键弱点在于用于诱导的方法 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 当前基于碰撞的碎片方法会以复杂的方式丢失。另一种选择 称为电子捕获解离 (ECD) 的碎裂方法已被广泛采用，可产生 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 的根本限制是难以提供足够的低能电子 有效地片段化肽。我们已经发现了如何使用精心雕刻的磁场和热 产生电子的灯丝，用于限制离子飞行路径中的大量电子。这可以是 适用于任何常见的串联质谱仪，无需改变现有的离子光学器件，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肽——最常见的工作流程 用于质谱分析。这种低碎片效率限制了灵敏度，这已被证明是 质谱行业采用这种强大方法的主要障碍。重点关注的是 第一阶段 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 通过磁场到窄光束来证明这个概念的可行性。目前反应时间为1-2 微秒。我们一期的可行性问题是至少能有效增加碎片化吗 通过短暂停止 ECD 细胞中的肽离子，可以实现两倍的效果，而不会因静电而造成显着损失 散射。此外，设计必须保留兼容所需的亚毫秒速度 当前前端 HPLC 和离子淌度分离与质谱仪一起用于复杂样品。 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到静电透镜短暂地停止它们的飞行。 可行性证明和经过验证的概念演示（第二阶段）对于吸引专业人士至关重要 仪器制造商进一步开发我们的 ECD 技术并将其商业化，以供大规模使用 光谱仪产品。成功还将展示我们的技术如何能够更好地碎片化 通过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分析物 碎片药物/代谢物。采用我们的技术将加速许多 NIH 的能力 研究人员探索疾病机制并确定诊断/治疗生物标志物 准确性和更快的速度，同时减少复杂生物样品中的错误识别。

项目成果

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

• DOI: 10.1021/acs.analchem.1c04870
• 发表时间: 2022-03-08
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Shaw JB;Cooper-Shepherd DA;Hewitt D;Wildgoose JL;Beckman JS;Langridge JI;Voinov VG
• 通讯作者: Voinov VG