Multiplexed, Single Molecule Protein Identification for Single Cell Proteomics

点击翻译按钮获取中文摘要

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.