National Center for Quantitative Biology of Complex Systems

项目总结--研发1 我们将这一研发引向改进生物分子表征的方向。理由很简单：你不能量化 你无法描述的东西！串联质谱仪(MS/MS)--表征的中心方法 生物分子与MS，通常是通过碰撞激活实现的。但是，此过程通常会失败，当 多肽前体含有抑制随机骨架质子化的氨基酸和通过 比酰胺键裂解所涉及的能量更低的途径。除了这些限制之外，还有几个 生物分子类-O-糖肽、糖胺多聚糖和修饰核酸，仅举几例- 优先以负模式电离，因此极难与电流排序 技术。为了获得这些具有挑战性的生物分子，全球各地的研究人员都转向了电子 转移解离(ETD)。现在是蛋白质组技术的中流砥柱，ETD使多肽阳离子与小分子 分子阴离子。也就是说，未来在生物分子表征方面的突破将需要关键的进展 在ETD技术方面。首先，ETD反应速度现在比(每次扫描~60-90毫秒)长一个数量级 基于碰撞的方法，限制了ETD在要求高吞吐量和 敏感度高。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(&gt；1,000 m/z)。这阻碍了ETD在许多应用中的使用，特别是 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，一个 ETD的变体--负电子转移解离(NETD)--具有巨大的潜力 酸性生物分子，但不是广泛可用的，因为还没有一种离子源可以产生高 进行反应所需试剂阳离子的助熔剂。在这里，我们利用我们深厚的历史和专业知识 ETD提出了创造性的、可出口的技术，以弥补这些关键的瓶颈。

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.