Photo-Fragmentation Methods for Single-Molecule Protein Sequencing by Nanopore Mass Spectrometry

项目总结 单分子蛋白质测序技术将彻底改变我们研究生物学的能力 以及诊断和治疗疾病。质谱学具有识别氨基酸的独特能力。 但目前的仪器远远达不到单分子的水平 灵敏度极限。另一方面，纳米孔传感器在单分子上工作，自然地 保持生物聚合物的顺序，但它们不能区分不同的氨基 酸。我们设想了一种蛋白质测序技术，在这种技术中，一种新的基于纳米孔的离子源 解决了当前质谱学的缺点。首先变性的基本思想是 蛋白质，然后驱动它通过一个纳米级的洞，迫使其组成的氨基酸进入 顺序排列，然后通过光碎裂将聚合物分解成单独的单体，以及 最后将这些氨基酸送入质谱仪，在那里它们可以通过它们的 质荷比。 我们最近展示了一种纳米孔离子源，它可以输送去溶的氨基酸离子。 从水溶液中直接进入质谱仪的高真空部分。纳米孔 因此，离子源绕过了传统电喷雾固有的样品损失机制 电离(ESI)，将带电的液滴喷射到散射离子的背景气体中 并降低了它们的传播速度。此外，我们最近设计了一种质谱仪， 展示了我们测序所需的两项新功能：1)同时测量 具有不同质量的离子以及2)以低于100 ns的到达时间标记每个离子的能力 时间分辨率。我们的排序策略的主要未经证实的部分是一种方法，用于 在纳米孔离子源内将多肽(即聚合物)转化为单独的氨基酸。 该项目将研究在纳米孔离子内用光碎裂多肽的可行性。 消息来源。我们寻求将多肽分割成独立但完整的氨基酸。紫外光与 200 nm附近的波长很有希望用于这一目的，因为它被 因为单个光子携带的能量足以引起多肽键的断裂 邦德。通过这个项目的目标，我们将衡量速度和选择性 不同波长的光会断裂多肽键，我们将把这一知识应用于 展示我们的纳米孔离子源中的多肽碎裂。

PROJECT SUMMARY A single molecule protein sequencing technology would revolutionize our ability to study biology and to diagnose and treat disease. Mass spectrometry has a unique ability to identify amino acids by their mass-to-charge ratios, but current instruments fall well short of the single-molecule sensitivity limit. On the other hand, nanopore sensors operate on single molecules and naturally preserve the sequential ordering of a biopolymer, but they cannot discriminate the different amino acids. We envision a protein sequencing technology in which a new, nanopore-based ion source addresses the current shortcomings of mass spectrometry. The basic idea to first denature the protein, then drive it through a nanoscale hole that compels its constituent amino acids to pass in sequential order, then break the polymer into separate monomers by photo-fragmentation, and finally deliver those amino acids into a mass spectrometer where they can be identified by their mass-to-charge ratios. We recently demonstrated a nanopore ion source that can deliver desolvated amino acid ions directly into the high vacuum part of a mass spectrometer from aqueous solution. The nanopore ion source thus circumvents the sample loss mechanisms inherent to conventional electrospray ionization (ESI), where charged droplets are sprayed into a background gas that scatters ions and degrades their transmission. Furthermore, we recently designed a mass spectrometer that exhibits two new capabilities we need for sequencing: 1) an ability to simultaneously measure ions with different masses and 2) an ability to tag each ion with an arrival time with sub-100 ns temporal resolution. The main unproven part of our sequencing strategy is a method for transforming peptides (i.e., polymers) into separate amino acids inside the nanopore ion source. This project will investigate the feasibility of fragmenting peptides with light within a nanopore ion source. We seek to fragment peptides into separate but intact amino acids. Ultraviolet light with a wavelength near 200 nm is promising for this purpose because it is selectively absorbed by peptide bonds and because a single photon carries enough energy to induce scission of that bond. Through the Aims of this project, we will measure the speed and selectivity with which different wavelengths of light cleave the peptide bond, and we will apply that knowledge to demonstrate peptide fragmentation within our nanopore ion source.