Dual Electron-Based Fragmentation with Ion Mobility to Advance Native Top-Down Proteomics

基于双电子的断裂和离子淌度以推进天然自上而下的蛋白质组学

• 批准号: 10009626
• 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: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009626
• 关键词: Address; Adoption; Affect; Alcohol dehydrogenase; Antibodies; Arthritis; Aspartate; Behavior; Biological; Biological Products; Businesses; Capsid; Cardiovascular Diseases; Cell Separation; Cell physiology; Cells; Charge; Communities; Complex; Computer software; Congestive; Development; Devices; Diabetes Mellitus; Diagnosis; Dimensions; Disease; Dissociation; Electron Transport; Electronics; Electrons; Erythrocytes; Family; Feasibility Studies; Filament; Future; Goals; Heart Diseases; Hemoglobin; Hour; Human; Inflammation; Ions; Isoleucine; Laboratories; Leucine; Location; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Methodology; Methods; Modernization; Multiprotein Complexes; Nerve Degeneration; Noble Gases; Pattern; Peptides; Periodicity; Phase; Physiologic pulse; Post-Translational Protein Processing; Problem Solving; Process; Protein Analysis; Protein Fragment; Proteins; Proteomics; Research Personnel; Resolution; Side; Small Business Innovation Research Grant; Speed; Stream; Technology; Testing; Tissues; Travel; Trypsin; United States National Institutes of Health; Variant; Vendor; Viral; Water; Work; base; beta-Aspartate; cost effective; design; flexibility; fly; improved; instrument; instrumentation; interest; ion mobility; macromolecule; mass spectrometer; operation; phase 1 study; preservation; protein complex; prototype; software development; success; tool; transmission process

The identification and quantification of biological macromolecules remain challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation lies in the methods used to induce fragmentation. The reliance in particular on collision-induced dissociation (CID) has limited such analyses to bottom-up workflows of trypsin-digested peptides of 10-30 residues. At e-MSion, we have developed an efficient electron-fragmentation technology called ExD now co-marketed with Agilent for their family of Q-TOFs and with Thermo for their QE Orbitraps. We succeeded with our phase I feasibility question to raise the fragmentation efficiency for doubly charged peptides from 1-3% to approaching 20%. This makes our ExD technology practical for peptide characterization and PTM localization in bottom up workflows -- the bread and butter for most proteomics laboratories. What has really captured the interest of the biopharma and the top-down communities in the past year is the exceptional sequence coverage of native proteins we obtain with the same ExD cell. The resulting spectra are less congested than those obtained with ETD/UVPD/CID fragmentation methodologies and it works for larger macromolecular protein complexes than has ever been possible before. Even with our simpler fragmentation patterns, the spectral congestion from proteoforms greater than ~30 kDa becomes too complex for many fragments to be distinguished even the highest resolution mass spectrometers. Our ExD technology is also faster than all other electron-based fragmentation methods. This speed allows entire proteins to be sequenced even after Ion Mobility Separations (IMS), which allows for spectra to be better resolved by adding a fourth dimension of resolution. Because of this unique capability, Waters recently purchased a prototype of our ExD cell adapted to fit at the exit of the IMS in their Synapt G2 mass spectrometer. Shortly after installation, we were able to sequence hemoglobin variants from native tetramers directly sprayed from human red blood cell lysates, FAB antibody subunits, and alcohol dehydrogenase (150 kDa). Some complexes such as GroEL and viral capsids still resist dissociation. We propose to overcome the challenges of both spectral congestion and dissociation of large native complexes by utilizing dual ExD cells with IMS. We will optimize the entrance-ExD cell to dissociate native protein complexes and use the exit- ExD cell to further fragment IMS-resolved subunits. We will develop the control electronics and software needed to coordinate the behavior of the two ExD cells with the IMS operation. Success will make possible characterization of larger proteoforms by top-down native proteomics than possible before. The adoption of our technology offers an extremely cost-effective solution that will accelerate the ability of many NIH investigators to probe disease mechanisms by characterizing complex macromolecules under native conditions with increased accuracy, speed, and fewer misidentifications.

尽管主要 现代质谱仪的速度，分辨率和质量准确性的进步。一个关键的弱点 当前的仪器在于用于诱导碎片化的方法。特别依赖 碰撞引起的解离（CID）的这种分析限制在胰蛋白酶消化的自下而上的工作流程 10-30种残基的肽。在E-MSION，我们开发了一种有效的电子碎片技术 现在被称为EXD，现在与Agilent共同营销了他们的Q-TOF家族，并与Thermo for He QE共同营销 轨道。我们在阶段I的可行性问题上取得了成功，以提高双重分裂效率 带电的肽从1-3％到20％。这使我们对肽的EXD技术实用 自下而上的工作流程中的表征和PTM定位 - 大多数蛋白质组学的面包和黄油 实验室。真正吸引了生物制药和自上而下社区的兴趣 过去一年是我们使用相同EXD细胞获得的天然蛋白质的特殊序列覆盖率。这 与ETD/UVPD/CID碎片方法获得的光谱相比 它适用于比以往任何时候都更大的大分子蛋白复合物。即使有我们的 更简单的碎裂模式，大于〜30 kDa的蛋白质的光谱充血也变得如此 复杂的许多片段甚至可以区分最高分辨率的质谱仪。我们的exd 技术的速度也比所有其他基于电子的碎片方法都要快。这种速度使整个 即使在离子迁移率分离（IMS）之后，也要测序的蛋白质，这使光谱变得更好 通过添加分辨率的第四维来解决。由于这种独特的能力，沃特斯最近 购买了我们的EXD细胞的原型 光谱仪。安装后不久，我们能够对天然四聚体的血红蛋白变体进行测序 直接从人类红细胞裂解物，晶圆厂抗体亚基和酒精脱氢酶喷涂（150 KDA）。一些复合物（例如凹槽和病毒式衣壳）仍然抵抗解离。我们建议克服 光谱充血和大型天然复合物的分离挑战通过使用双EXD细胞 与IMS。我们将优化入口细胞以解离天然蛋白质复合物并使用出口 EXD细胞以进一步碎片IMS分辨亚基。我们将开发控制电子和软件 需要与IMS操作协调两个EXD单元的行为。成功将成为可能 自上而下的天然蛋白质组学对较大蛋白质的表征比以前可能。采用 我们的技术提供了一种极具成本效益的解决方案，可以加快许多NIH的能力 研究人员通过表征天然的复杂大分子来探测疾病机制 准确性，速度和较少误解的条件。