Multiplexed, Single Molecule Protein Identification for Single Cell Proteomics

描述（由申请人提供）：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。这将通过结合“自下而上”的方法来实现蛋白质组学通常采用质谱与单分子荧光显微镜和微阵列平台。单个蛋白质分子将在经化学修饰以捕获所得肽的微米级腔室中胰蛋白酶化（Aim 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，单分子荧光成像可用于从单个胰蛋白酶肽获得足够的序列信息，以鉴定单个蛋白质分子（目的2）。单分子灵敏度和 由该技术提供的数字定量将用于在各种实验环境中测量单细胞中的蛋白质丰度。特别是，具有集成蛋白质识别阵列的微流体装置将与激光捕获显微切割连接，以从胶质母细胞瘤样品中提取单个细胞。个体癌细胞的大规模蛋白质组学表征将提供肿瘤异质性的前所未有的视角，这是有效治疗多种形式癌症的关键障碍。 公共卫生相关性：癌症和宿主-病原体系统中的异质结对病变组织的常规批量研究构成了严重挑战。通过对来自单个细胞的大部分蛋白质进行表征和定量，该项目将显著提高我们理解这些复杂系统、分析临床样本并最终设计更有效治疗方法的能力。

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. PUBLIC HEALTH RELEVANCE: Heterogeneity in cancer and host-pathogen systems poses a serious challenge to conventional bulk studies of diseased tissue. By enabling the characterization and quantification of a large fraction of proteins from individual cells, this proect will significantly enhance our ability to understand these complex systems, analyze clinical samples, and ultimately design more effective treatments.