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

生物大分子的鉴定和定量仍然具有挑战性，尽管主要 现代质谱仪在速度、分辨率和质量精度方面的进步。一个关键弱点是， 目前的手段在于用于诱导碎裂的方法。特别是依赖于 碰撞诱导解离（CID）将这种分析限制于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，我们开发了一种有效的电子碎裂技术， 名为ExD，现在与Agilent共同销售其Q-TOF系列，与Thermo共同销售其QE 轨道探测器我们成功地解决了第一阶段的可行性问题，将碎片效率提高了一倍， 带电荷的肽从1-3%到接近20%。这使得我们的ExD技术对肽具有实用性 自下而上工作流程中的特征化和PTM定位--大多数蛋白质组学的基础 laboratories.真正吸引生物制药和自上而下的社区兴趣的是 在过去的一年中，我们用相同的ExD细胞获得的天然蛋白质的序列覆盖率非常高。的 得到的光谱比用ETD/UVPD/CID裂解方法得到的光谱不那么拥挤 它对更大的大分子蛋白质复合物的作用比以往任何时候都要大。即使我们 更简单的片段化模式，来自大于~30 kDa的蛋白质型的光谱拥塞也变得 即使是最高分辨率的质谱仪也难以区分许多碎片。我们的前任 技术也比所有其他基于电子的碎裂方法更快。这个速度让整个 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 通过增加分辨率的第四维来解决。由于这种独特的能力，沃茨最近 我购买了一个我们的ExD细胞的原型，适合在IMS的出口，在他们的突触G2质量 光谱仪安装后不久，我们就能够从天然四聚体中测序血红蛋白变体 直接从人红细胞裂解物、FAB抗体亚单位和醇脱氢酶（150 kDa）。一些复合物如GroEL和病毒衣壳仍然抵抗解离。我们建议克服 通过利用双ExD细胞的光谱拥塞和大的天然复合物的解离的挑战 关于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细胞以进一步片段化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小区的行为与IMS操作。成功将使 通过自上而下的天然蛋白质组学表征比以前可能的更大的蛋白质型。通过 我们的技术提供了一个极具成本效益的解决方案，将加快许多国家卫生研究院的能力， 研究人员通过在天然条件下表征复杂的大分子来探测疾病机制 提高了准确性、速度和更少的错误识别。

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.