National Center for Quantitative Biology of Complex Systems

项目概要- TR&D 1 我们将此TR&D导向改进的生物分子表征。理由很简单：你不能量化 你无法描述的东西串联质谱法（MS/MS），表征 生物分子与MS的结合通常通过碰撞活化来实现。然而，这个过程经常失败， 肽前体含有抑制随机骨架质子化的氨基酸和通过质子化解离的PTM。 一种比酰胺键断裂所涉及的能量更低的途径。除了这些限制， 生物分子类别-O-糖肽、糖胺聚糖和修饰的核酸，仅举几例- 优先在负模式下工作，因此极难用电流定序 技术.为了获得这些具有挑战性的生物分子，地球仪的研究人员转向了电子 转移解离（ETD）。现在是蛋白质组学技术的支柱，ETD将肽阳离子与小分子 分子阴离子。也就是说，未来生物分子表征的突破将需要关键的进步 在ETD技术。首先，ETD反应速率现在比传统的ETD反应速率长一个数量级（每次扫描约60-90 ms）， 基于冲突的方法，限制了ETD在要求高吞吐量的现代时代可能产生的影响， 高灵敏度。其次，将肽序列分配给ETD扫描的成功率受到前体的影响， 具有低电荷密度（> 1，000 m/z）。这阻碍了ETD在许多应用中的使用，特别是那些 涉及增加质量而不增加电荷的大生物分子或PTM（例如，糖基化）。最后， ETD的变体-负电子转移解离（NETD）-具有巨大的潜力，可以获得 酸性生物分子，但由于还没有可以产生高浓度的离子源， 进行反应所需的试剂阳离子的通量。在这里，我们利用我们深厚的历史和专业知识， ETD将提出创新的、可出口的技术，以解决这些关键瓶颈。

PROJECT SUMMARY – TR&D 1 We direct this TR&D toward improved biomolecule characterization. The rationale is simple: you cannot quantify what you cannot characterize! Tandem mass spectrometry (MS/MS), the central approach for characterizing biomolecules with MS, is typically achieved by collisional activation. This process often fails, however, when the peptide precursor contains amino acids that inhibit random backbone protonation and PTMs that dissociate by a lower energy pathway than that involved in the cleavage of the amide linkage. Beyond these limitations, several biomolecule classes – O-glycopeptides, glycosaminoglycans, and modified nucleic acids, to name a few – preferentially ionize in the negative mode and are therefore extremely difficult to sequence with current technologies. To access these challenging biomolecules, researchers across the globe have turned to electron transfer dissociation (ETD). Now a mainstay in proteomic technology, ETD reacts peptide cations with small molecule anions. That said, future breakthroughs in biomolecule characterization will require key advancements in ETD technology. First, ETD reaction rates are now an order of magnitude longer (~60–90 ms per scan) than collision-based methods, limiting the impact ETD can have in a modern era that demands high throughput and high sensitivity. Second, the success rate of assigning peptide sequence to ETD scans suffers for precursors with low charge density (>1,000 m/z). That impedes ETD’s use in a number of applications, especially those involving large biomolecules or PTMs that add mass without adding charge (e.g., glycosylation). And finally, a variant of ETD – negative electron transfer dissociation (NETD) – has tremendous potential to allow access to acidic biomolecules, but is not widely available because there is not yet an ion source that can produce a high flux of the reagent cations needed to conduct the reaction. Here we leverage our deep history and expertise in ETD to propose creative, exportable technologies that remedy these crucial bottlenecks.