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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21EB016980-02
Application #
8549924
Study Section
Special Emphasis Panel (ZRG1-CB-D (51))
Program Officer
Conroy, Richard
Project Start
2012-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$194,000
Indirect Cost
$72,750
Name
Columbia University (N.Y.)
Department
Biochemistry
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032