Multiplexed, Single Molecule Protein Identification for Single Cell Proteomics

单细胞蛋白质组学的多重、单分子蛋白质鉴定

• 批准号: 8549924
• 负责人: Peter Alan Sims
• 金额: $ 19.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8549924
• 关键词: Amino Acids; Antibodies; Aspartate; Base Pairing; Base Sequence; Biological Markers; Cells; Chimeric Proteins; Cleaved cell; Clinical; Color; Complex; Cysteine; DNA Sequence; Databases; Detection; Digestion; Endopeptidases; Fluorescence Microscopy; Gene Expression Profile; Genome; Genomics; Glass; Glioblastoma; Glutamates; Heterogeneity; Image; Individual; Label; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Medicine; Methods; Microfluidic Microchips; Microfluidics; Microscopy; Molecular Profiling; Monitor; Nucleic Acids; Optics; Pattern; Peptide Hydrolases; Peptides; Phenotype; Process; Protein Analysis; Proteins; Proteome; Proteomics; Reagent; Relative (related person); Research Personnel; Sampling; Site; Solid; Surface; System; Systems Analysis; Techniques; Technology; Tissues; Trypsin; analog; base; biological research; cancer cell; design; digital; effective therapy; fluorescence imaging; fluorescence microscope; fluorophore; laser capture microdissection; next generation sequencing; novel; novel strategies; pathogen; polydimethylsiloxane; seal; single cell analysis; single molecule; success; tool; tumor

DESCRIPTION (provided by applicant): The main objective of this project is to enable single cell proteomics by developing a novel, ultrasensitive technology for protein identification. This will be accomplished by combining the "bottom-up" approach to proteomics typically employed in mass spectrometry with single molecule fluorescence microscopy and a microfabricated array platform. Single protein molecules will be trypsinized in micron-scale chambers that are chemically modified to capture the resultant peptides (Aim 1). By applying amino acid-specific labels and monitoring the activity of amino acid-specific endopeptidases, single molecule fluorescence imaging can be used to obtain sufficient sequence information from individual tryptic peptides to identify a single protein molecule (Aim 2). The single molecule sensitivity and digital quantification afforded by this technique will be used to measure protein abundance in single cells in a variety of experimental contexts. In particular, a microfluidic device with an integrated protein identification array will be interfaced with laser capture microdissection to extract individual cells from glioblastoma samples. Large-scale proteomic characterization of individual cancer cells will provide an unprecedented view of tumor heterogeneity, a key obstacle to effectively treating many forms of cancer.

描述(申请人提供)：该项目的主要目标是通过开发一种新的、超灵敏的蛋白质鉴定技术来实现单细胞蛋白质组学。这将通过将通常用于质谱学的蛋白质组学的“自下而上”方法与单分子荧光显微镜和微制阵列平台相结合来实现。单个蛋白质分子将在微米级的小室中被胰酶消化，这些小室经过化学修饰以捕获生成的多肽(目标1)。通过应用氨基酸特异性标记和监测氨基酸特异性内肽酶的活性，单分子荧光成像可以从单个胰蛋白酶多肽中获得足够的序列信息，以识别单个蛋白质分子(目标2)。单分子灵敏度和 这项技术提供的数字量化将用于在各种实验环境中测量单个细胞中的蛋白质丰度。特别是，带有集成蛋白质鉴定阵列的微流控设备将与激光捕获显微切割相连接，以从胶质母细胞瘤样本中提取单个细胞。对单个癌细胞的大规模蛋白质组学表征将提供对肿瘤异质性的前所未有的看法，这是有效治疗多种形式癌症的关键障碍。