Long-read single-molecule protein sequencing on an array of unfoldase-coupled nanopores

在一系列解折叠酶偶联纳米孔上进行长读长单分子蛋白质测序

基本信息

批准号：
10708013
负责人：
Jeffrey Matthew Nivala
金额：
$ 61.2万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-21 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10708013
关键词：
26S proteasome Affinity Amino Acid Sequence Amino Acid Substitution Amino Acids Biochemical Bioinformatics Biological Cell Culture Techniques Cells Collaborations Combinatorics Complex Coupled DNA Data Databases Detection Development Devices Disease Engineering Escherichia coli Eukaryotic Cell Foundations Genome Grant Human Human Genome In Vitro Individual Label Length Malignant Neoplasms Mass Spectrum Analysis Methods Modeling Modification Molecular Motors Motor Motor Activity Movement N-terminal Nucleic Acids Nucleic acid sequencing Peptide Sequence Determination Peptides Performance Phenotype Pore Proteins Post-Translational Protein Processing Prokaryotic Cells Protein Isoforms Protein translocation Proteins Proteome Proteomics Qualifying Reagent Research Personnel Resolution Role Route Sampling Science Sequence Analysis Signal Transduction Structure Techniques Technology Technology Transfer Tertiary Protein Structure Testing Touch sensation Training Translating Variant base chemical conjugate dark matter detection method detector experience insight markov model model organism nanometer nanopore neural network protein aminoacid sequence research and development sensor sequencing platform single molecule technology platform tool transcriptome transcriptome sequencing unfoldase

项目摘要

SUMMARY We propose to develop the foundations of a platform for direct sequencing of native, full-length protein strands using unfoldase-coupled nanopore array technology. In principle, this technology could be used to identify protein primary sequence, in addition to certain post-translational modifications (PTMs) found in prokaryotic and eukaryotic cells, with single-molecule resolution. It is a foundational advance over existing and other next-gen proteomic technologies such as Edman degradation, mass spectrometry, fluorescent label approaches, and immunoaffinity-based methods that suffer from limitations in read length, throughput, sensitivity, labeling efficiency, and/or the availability of suitable affinity reagents. Nanopore sequencing of intact protein strands overcomes these limitations because the ~1 nanometer-long sensor directly interacts with the protein strand as it is linearly-driven through the pore by the unfoldase motor protein, manifesting sequence-specific ionic current signals. Thus, complete sequence analysis of native protein molecules can be achieved. This method is a natural technical extension of current nanopore sequencing platforms that use molecular motors to control movement of nucleic acid strands through nanopores in DNA/RNA sequencing. During the grant period, we will pursue three specific aims: 1) Establish baseline methods of controlled protein translocation through nanopore sensor arrays using unfoldase motors; 2) Develop computational and bioinformatic methods to translate raw nanopore signal data into protein sequence information (amino acid calling and PTM detection); and 3) Establish techniques for analysis of native proteins and proteomic samples. Our team of investigators is uniquely qualified to take on this project: i) We pioneered the analysis of full-length protein strands using unfoldase-coupled nanopore sensors and recently demonstrated that the Oxford Nanopore MinION nanopore array device can be used to directly detect peptide strands and resolve single amino acid substitutions (Nivala). ii) Co-investigators on this application have elucidated and exquisitely characterized the enzymatic mechanisms of unfoldase motor activity through in vitro biochemical, single-molecule, and structural studies (Martin), and have led the development of nanopore raw signal analyses for sequencing of nucleic acids, including direct RNA sequencing, genome and transcriptome-wide detection of modified bases, and assembly of a human genome using ultra-long DNA nanopore reads (Jain). iii) Collaborators will provide access to enabling nanopore technology platforms and expertise, including highly-parallel nanopore sensor arrays and customized nanopore proteins, and offer natural routes to technology transfer (Oxford Nanopore), contribute to characterization and comparison of project results to traditional analysis methods such as protein mass spectrometry (Guttman), and advise on compelling technological applications that will be enabled by successful execution of this project (Timp).

概括我们建议开发一个平台的基础，用于对天然全长蛋白质链进行直接测序使用非折叠酶耦合纳米孔阵列技术。原则上，该技术可用于识别蛋白质一级序列，以及原核生物中发现的某些翻译后修饰 (PTM) 和真核细胞，具有单分子分辨率。这是对现有和其他技术的根本性进步下一代蛋白质组技术，例如 Edman 降解、质谱、荧光标记方法和基于免疫亲和力的方法受到读长、吞吐量的限制，灵敏度、标记效率和/或合适的亲和试剂的可用性。完整的纳米孔测序蛋白质链克服了这些限制，因为约 1 纳米长的传感器直接与蛋白质链，因为它由解折叠酶马达蛋白线性驱动通过孔，表现出序列特异性离子电流信号。因此，可以对天然蛋白质分子进行完整的序列分析实现了。该方法是当前纳米孔测序平台的自然技术延伸，该平台使用分子马达在 DNA/RNA 测序中控制核酸链通过纳米孔的运动。在资助期间，我们将追求三个具体目标：1）建立受控蛋白质的基线方法使用去折叠酶马达通过纳米孔传感器阵列进行易位； 2）发展计算和将原始纳米孔信号数据转化为蛋白质序列信息（氨基酸呼叫和 PTM 检测）； 3) 建立天然蛋白质和蛋白质组样品的分析技术。我们的研究团队拥有承担该项目的独特资质： i) 我们率先使用解折叠酶耦合纳米孔传感器分析全长蛋白质链，最近证明Oxford Nanopore MinION纳米孔阵列装置可用于直接检测肽链并解析单个氨基酸取代（Nivala）。 ii) 该应用的共同研究者已经阐明并精确地表征了酶促通过体外生化、单分子和结构研究了解解折叠酶运动活性的机制（马丁），并领导了用于核酸测序的纳米孔原始信号分析的开发，包括直接 RNA 测序、修饰碱基的基因组和转录组范围检测以及组装使用超长 DNA 纳米孔读数分析人类基因组 (Jain)。 iii) 合作者将提供纳米孔技术平台和专业知识，包括高度并行的纳米孔传感器阵列和定制的纳米孔蛋白质，并提供自然途径技术转让（牛津纳米孔），有助于项目结果的表征和比较传统的分析方法，例如蛋白质质谱（Guttman），并提供令人信服的建议该项目的成功执行将实现技术应用（Timp）。