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: 8473887
• 负责人: JAMES J HAVRANEK
• 金额: $ 27.87万
• 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/8473887
• 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; Genomics; Goals; Imagery; Individual; Measures; Medical; Methionine; Methods; Molecular; Mutation; N-terminal; NanoGel; Natural regeneration; Nature; Nucleic Acids; 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; genome analysis; high throughput analysis; improved; inorganic phosphate; methyl group; mutant; next generation; next generation sequencing; 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.

描述（由申请人提供）：最近核酸测序能力的爆炸式增长导致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，以实现类似的蛋白质和肽的多路单分子测序。单分子检测的最新进展使这成为一个可行的目标。然而，与核酸测序相比，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。蛋白质设计和工程必须用来产生必要的分子试剂。我们设想的蛋白质测序的长期战略是在单分子上进行Edman降解。蛋白质工程将用于使天然存在的蛋白质对游离氨基酸具有内在亲和力和特异性，以作为肽中n端残基的序列特异性结合物。可视化将用单分子荧光显微镜进行。一种半胱氨酸蛋白酶将被用来去除末端氨基酸，以再生一个新的肽n端，用于后续的测序。现有的蛋白质可以识别翻译后修饰的蛋白质和20种典型氨基酸，这表明该方法可以应用于研究蛋白质组的翻译后状态和内容。具体目标是1)设计tRNA合成酶作为n端测序试剂，2)修饰半胱氨酸蛋白酶以去除已被Edman试剂修饰的n端氨基酸，3)设计一组三种蛋白质以实现磷酸化氨基酸的测序。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。