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.

尽管存在重大问题，生物大分子的识别和定量仍然具有挑战性 现代质谱仪在速度、分辨率和质量精度方面取得了进步。一个关键弱点是 当前的仪器在于用于诱导碎片的方法。尤其是依赖于 碰撞诱导解离 (CID) 将此类分析限制为胰蛋白酶消化的自下而上的工作流程 10-30个残基的肽。在 e-MSion，我们开发了一种高效的电子碎片技术 名为 ExD 的公司现在与安捷伦共同销售其 Q-TOF 系列，并与 Thermo 共同销售其 QE 轨道陷阱。我们成功解决了第一阶段的可行性问题，将碎片效率提高了一倍 带电肽从 1-3% 增加到接近 20%。这使得我们的 ExD 技术适用于肽 自下而上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。是什么真正引起了生物制药公司和自上而下社区的兴趣 去年我们使用相同的 ExD 细胞获得了天然蛋白质的出色序列覆盖率。这 所得光谱比使用 ETD/UVPD/CID 碎片方法获得的光谱更不拥挤 它适用于比以前更大的大分子蛋白质复合物。即使与我们的 更简单的碎片模式，来自大于 ~30 kDa 的蛋白质形式的光谱拥塞变得太 即使是最高分辨率的质谱仪，也很难区分许多碎片。我们的ExD 该技术也比所有其他基于电子的碎片方法更快。这个速度允许整个 即使在离子淌度分离 (IMS) 后也能对蛋白质进行测序，从而获得更好的光谱 通过添加第四维分辨率来解决。由于这种独特的能力，沃特世最近 购买了我们的 ExD 细胞原型，适合安装在 Synapt G2 质量中的 IMS 出口处 光谱仪。安装后不久，我们就能够对天然四聚体的血红蛋白变体进行测序 直接从人红细胞裂解物、FAB 抗体亚基和乙醇脱氢酶（150 kDa）。一些复合物（例如 GroEL 和病毒衣壳）仍能抵抗解离。我们建议克服 利用双 ExD 细胞应对光谱拥塞和大型天然复合物解离的挑战 与IMS。我们将优化入口 ExD 细胞以解离天然蛋白质复合物并使用出口 ExD 细胞进一步裂解 IMS 解析的亚基。我们将开发控制电子设备和软件 需要协调两个 ExD 单元的行为与 IMS 操作。成功将让一切成为可能 通过自上而下的天然蛋白质组学来表征比以前更大的蛋白质形式。通过 我们的技术提供了一种极具成本效益的解决方案，将加速许多 NIH 的能力 研究人员通过表征天然条件下的复杂大分子来探索疾病机制 提高了准确性、速度并减少了错误识别。