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:
- 金额:$ 43.86万
- 依托单位:
- 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
- 来源:
- 关键词:AddressAmino AcidsBiologyBiopolymersChargeChemicalsDevelopmentDiagnosisDiagnosticDiameterDiseaseDissociationDoseElectrospray IonizationEnvironmentEvaluationExhibitsGasesGovernmentIndividualIonsKnowledgeLasersLightMass Spectrum AnalysisMeasuresMethodsModelingMonitorOpticsPeptide FragmentsPeptide Sequence DeterminationPeptidesPersonsPhotonsPolymersProbabilityProcessProtein FragmentProteinsProteomicsSamplingSchemeSpecificitySpeedTechnologyTimeUltraviolet RaysVacuumVertebral columnWaterabsorptionaqueousdensitydesigndetectorfallsinstrumention sourceionizationirradiationmass spectrometermonomernanoporenanoscalepreservationsensorsingle moleculesynthetic peptidetemporal measurementtransmission process
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.
项目摘要
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
诊断和治疗疾病。质谱分析法具有独特的鉴定氨基酸的能力
但目前的仪器还远远达不到单分子
灵敏度极限另一方面,纳米孔传感器对单分子进行操作,
保留生物聚合物的顺序,但它们不能区分不同的氨基
acids.我们设想了一种蛋白质测序技术,其中一种新的基于纳米孔的离子源
解决了质谱法的当前缺点。基本的想法是首先变性
蛋白质,然后驱动它通过一个纳米级的孔,迫使其组成氨基酸通过
按顺序排列,然后通过光断裂将聚合物断裂成单独的单体,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,
从水溶液直接进入质谱仪的高真空部分。纳米孔
离子源因此避免了传统电喷雾固有的样品损失机制
电离(ESI),其中带电液滴被喷射到散射离子的背景气体中
并降低其传输。此外,我们最近设计了一个质谱仪,
展示了我们测序所需的两种新能力:1)同时测量
具有不同质量的离子和2)以小于100 ns的到达时间标记每个离子的能力
时间分辨率我们的测序策略的主要未经证实的部分是一种方法,
转化肽(即,聚合物)转化为纳米孔离子源内的单独氨基酸。
这个项目将研究在纳米孔离子内用光使肽片段化的可行性
源头我们试图将肽片段化为独立但完整的氨基酸。的紫外光
接近200 nm的波长对于该目的是有希望的,因为它被
因为单个光子携带足够的能量来诱导肽键的断裂,
邦德通过本项目的目标,我们将衡量速度和选择性,
不同波长的光切割肽键,我们将把这些知识应用到
证明了我们的纳米孔离子源内的肽片段化。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Derek Stein其他文献
Derek Stein的其他文献
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