A MOLECULAR TOOLKIT FOR SINGLE-MOLECULE PROTEIN SEQUENCING

用于单分子蛋白质测序的分子工具包

• 批准号: 8275377
• 负责人: JAMES J HAVRANEK
• 金额: $ 28.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8275377
• 关键词: Acids; Affinity; Amino Acid Sequence; Amino Acids; Amino Acyl-tRNA Synthetases; Base Sequence; Benign; Binding; Binding Sites; Biological Assay; Biological Markers; Buffers; Characteristics; Chemicals; Cleaved cell; Coupled; Cysteine Protease; Data; Detection; Disease; Engineering; Enzymes; Equipment and supply inventories; Explosion; Fluorescence Microscopy; Genome; Genomics; Goals; Imagery; Individual; Measures; Medical; Methionine; Methods; Molecular; Mutation; N-terminal; NanoGel; Natural regeneration; Nature; Nucleic acid sequencing; Outcome; Peptide Hydrolases; Peptide Sequence Determination; Peptides; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotyrosine; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Engineering; Proteins; Proteome; Proteomics; RS protein; Reagent; Relative (related person); Research; Resolution; Resort; Sampling; Set protein; Specificity; Structural Models; Structure; Technology; Temperature; Tertiary Protein Structure; Threonine; Tissues; Variant; analog; aqueous; base; cell type; comparative; design; directed evolution; engineering design; high throughput analysis; improved; inorganic phosphate; methyl group; mutant; next generation; novel; phenylisothiocyanate; protein aminoacid sequence; single molecule; tyrosine O-sulfate

DESCRIPTION (provided by applicant): The recent explosion of nucleic acid sequencing capacity has given rise to routine high-throughput analysis of transcriptional and genomic variation. The goal of the proposed research is to create a toolkit of molecular reagents to enable similarly multiplexed single molecule sequencing of proteins and peptides. Recent advances in single molecule detection make this a feasible goal. However, in contrast to nucleic acid sequencing, nature has not provided us with suitable enzymes and amino acid-identifying proteins to perform this analysis. Protein design and engineering must be utilized to generate the necessary molecular reagents. The long-range strategy we envision for protein sequencing is to perform Edman degradation on single molecules. Protein engineering will be used to adapt naturally occurring proteins with intrinsic affinity and specificity for free amino acids to serve s sequence-specific binders of N-terminal residues in peptide. Visualization will be performed with single molecule fluorescence microscopy. A cysteine protease will be engineered to remove terminal amino acids to regenerate a new peptide N-terminus for subsequent rounds of sequencing. The ready availability of proteins that recognize post-translationally modified as well as the twenty canonical amino acids suggests that this method can be applied to study the post-translational state, as well as the content, of the proteome. The specific aims are 1) to engineer tRNA synthetases to serve as N-terminal sequencing reagents, 2) to modify a cysteine protease to remove N-terminal amino acids that have been modified with the Edman reagent, 3) to engineer a set of three proteins to enable the sequencing of phosphorylated amino acids. Preliminary results demonstrate that these aims are feasible. Completion of this research will move next-generation protein sequencing much closer to being a reality. PUBLIC HEALTH RELEVANCE: The complete and quantitative analysis of proteomic inventory is crucial for identifying and measuring biomarkers, those proteins whose levels differ between diseased and unaffected tissues. The ability to identify medical problems as early as possible improves outcomes. The ability to determine phosphorylation state in this analysis can make even finer distinctions between the biologically relevant states of different samples. The molecules we propose to engineer for protein sequencing could enable proteomic analysis that has excellent dynamic range, is inherently quantitative, and is sensitive to amino acid phosphorylation state.

描述（由申请人提供）：最近核酸测序能力的爆炸式增长已经引起了转录和基因组变异的常规高通量分析。拟议研究的目标是创建一个分子试剂工具包，以实现蛋白质和肽的类似多重单分子测序。单分子检测的最新进展使其成为一个可行的目标。然而，与核酸测序相反，大自然并没有为我们提供合适的酶和氨基酸识别蛋白来进行这种分析。必须利用蛋白质设计和工程来产生必要的分子试剂。我们设想的蛋白质测序的长期策略是对单个分子进行Edman降解。蛋白质工程将用于改造对游离氨基酸具有内在亲和力和特异性的天然存在的蛋白质，以作为肽中N-末端残基的序列特异性结合剂。将使用单分子荧光显微镜进行可视化。半胱氨酸蛋白酶将被工程化以去除末端氨基酸，以再生新的肽N-末端用于随后的几轮测序。识别翻译后修饰的蛋白质以及20种典型氨基酸的现成可用性表明，这种方法可以应用于研究蛋白质组的翻译后状态以及内容。具体目标是1）工程化tRNA合成酶以用作N-末端测序试剂，2）修饰半胱氨酸蛋白酶以去除已经用Edman试剂修饰的N-末端氨基酸，3）工程化一组三种蛋白质以使得能够对磷酸化氨基酸进行测序。初步结果表明，这些目标是可行的。这项研究的完成将使下一代蛋白质测序更接近现实。 公共卫生关系：蛋白质组库存的完整和定量分析对于识别和测量生物标志物至关重要，这些蛋白质的水平在患病和未受影响的组织之间存在差异。尽早发现医疗问题的能力可以改善结果。在该分析中确定磷酸化状态的能力可以在不同样品的生物学相关状态之间进行更精细的区分。我们提出的用于蛋白质测序的分子可以使蛋白质组学分析具有良好的动态范围，本质上是定量的，并且对氨基酸磷酸化状态敏感。