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

• 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
• 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

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).

总结 我们建议开发一个平台的基础，直接测序的天然，全长蛋白质链 使用展开酶耦合纳米孔阵列技术。原则上，这项技术可以用来识别 蛋白质一级序列，除了在原核生物中发现的某些翻译后修饰（PTM）之外， 和真核细胞，用单分子分辨率。这是对现有和其他技术的基础性进步 下一代蛋白质组学技术，如Edman降解、质谱、荧光标记 方法和基于免疫亲和性的方法受到读长，通量， 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~1纳米长的传感器直接与 当它被解折叠酶马达蛋白线性驱动通过孔时， 序列特异性离子电流信号。因此，天然蛋白质分子的完整序列分析可以被实施。 办妥了一批该方法是当前纳米孔测序平台的自然技术延伸， 分子马达用于在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 使用解折叠酶马达通过纳米孔传感器阵列移位; 2）开发计算和 将原始纳米孔信号数据翻译成蛋白质序列信息（氨基酸）的生物信息学方法 调用和PTM检测）;和3）建立用于分析天然蛋白质和蛋白质组样品的技术。 我们的研究团队是唯一有资格承担这个项目： i）我们率先使用解折叠酶偶联的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 肽链和解析单个氨基酸取代（Nivala）。 ii）关于该应用的共同研究者已经阐明并精细地表征了酶促反应。 通过体外生化、单分子和结构研究解折叠酶运动活性的机制 （Martin），并且已经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，基因组和转录组范围内的修饰碱基检测， 使用超长DNA纳米孔读取（Jain）的人类基因组。 iii）合作者将提供使能纳米孔技术平台和专业知识，包括 高度平行的纳米孔传感器阵列和定制的纳米孔蛋白质，并提供自然的途径， 技术转让（Oxford Nanopore），有助于项目结果表征和比较， 传统的分析方法，如蛋白质质谱法（古特曼），并建议强制 技术应用，将通过成功执行该项目（Timp）。