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

纳米孔质谱单分子蛋白质测序的光断裂方法

基本信息

批准号：
10644378
负责人：
Derek Stein
金额：
$ 43.86万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-24 至 2025-04-30
项目状态：
未结题

项目摘要

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.

项目概要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。质谱法具有独特的氨基酸鉴定能力通过它们的质荷比，但目前的仪器远远达不到单分子灵敏度极限。另一方面，纳米孔传感器在单分子上运行，自然保留生物聚合物的顺序，但它们不能区分不同的氨基酸。我们设想一种蛋白质测序技术，其中一种新的、基于纳米孔的离子源解决了目前质谱分析的缺点。首先将其变性的基本思想蛋白质，然后驱动它通过纳米级的孔，迫使其组成氨基酸进入按顺序排列，然后通过光断裂将聚合物分解成单独的单体，并且最后将这些氨基酸送入质谱仪，在那里可以通过它们的氨基酸来识别它们质荷比。我们最近展示了一种纳米孔离子源，可以提供去溶剂化的氨基酸离子从水溶液直接进入质谱仪的高真空部分。纳米孔因此，离子源避免了传统电喷雾固有的样品损失机制电离 (ESI)，将带电液滴喷射到散射离子的背景气体中并降低其传输性能。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）同时测量的能力具有不同质量的离子，2) 能够以低于 100 ns 的到达时间标记每个离子时间分辨率。我们的测序策略中未经证实的主要部分是一种方法将肽（即聚合物）转化为纳米孔离子源内的单独氨基酸。该项目将研究在纳米孔离子内用光裂解肽的可行性来源。我们寻求将肽片段化成单独但完整的氨基酸。紫外线与接近 200 nm 的波长有希望用于此目的，因为它被选择性地吸收肽键，并且因为单个光子携带足够的能量来诱导肽键的断裂纽带。通过该项目的目标，我们将衡量其速度和选择性不同波长的光会裂解肽键，我们将应用这些知识证明我们的纳米孔离子源内的肽碎片。