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.

生物大分子的鉴定和量化仍然具有挑战性，尽管 现代质谱仪在速度、分辨率和质量精度方面的进展。的一个主要弱点 目前的手段在于用于诱导碎片化的方法。尤其是对 碰撞诱导解离(CID)将这种分析限制在胰酶消化的自下而上的工作流程中 10-30个残基的多肽。在e-MSion，我们开发了一种高效的电子碎裂技术 名为EXD的产品现已与安捷伦共同销售其Q-TOF系列产品，并与Thermo共同销售其QE产品 轨道陷阱。我们成功地解决了第一阶段的可行性问题，将碎片化效率提高了一倍 带电多肽从1-3%到接近20%。这使得我们的EXD技术适用于多肽 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。真正引起生物制药和自上而下社区的兴趣的是什么 过去的一年是我们用相同的EXD细胞获得的天然蛋白质的特殊序列覆盖率。这个 得到的光谱比ETD/UVPD/CID碎片方法获得的光谱不那么拥挤 而且，它可以处理比以往任何时候都更大的大分子蛋白质复合体。即使是在我们的 更简单的碎裂模式，大于~30 kDa的蛋白质形式的光谱拥堵也会变得 对于许多碎片来说，即使是最高分辨率的质谱仪也难以分辨。我们的EXD 这项技术也比所有其他基于电子的碎裂方法更快。这一速度使整个 即使在离子迁移率分离(IMS)之后也可以对蛋白质进行测序，这使得光谱更好 通过增加第四个维度的分辨率来解决。由于这一独特的能力，Waters最近 购买了我们的EXD细胞原型，适用于其Synapt G2 MASS中的IMS出口处 分光计。在安装后不久，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抗体亚基和酒精脱氢酶(150)喷洒 KDA)。一些复合体，如GroEL和病毒衣壳，仍然抵抗解离。我们建议克服 利用双EXD池应对光谱拥塞和大分子天然络合物解离的挑战 与IMS合作。我们将优化Entry-Exd细胞以分离天然蛋白质复合体，并利用Exit-Exd细胞 EXD单元格以进一步分段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。成功将使一切成为可能 通过自上而下的天然蛋白质组学比以前可能的更大的蛋白质形式的特征。采用了 我们的技术提供了极具成本效益的解决方案，将加速许多NIH的能力 研究人员将通过表征天然状态下的复杂大分子来探索疾病机制 具有更高的精确度、速度和更少的误识别。