Most biological processes are executed by proteins, but no method currently exists to accurately measure protein abundance and post-translational state proteome-wide. To redress this deficiency, we propose Digital Analysis of Proteins by End Sequencing (DAPES), a method that sequences many individual peptide molecules in parallel using Edman degradation. DAPES will be cost-effective, highly sensitive, and quantitative. DAPES is based on two innovations - 1) the use of dye-labeled antibodies to inexpensively and robustly detect single peptide molecules;and 2) a strategy that uses a universal set of ~20 antibodies to sequence peptide molecules. Our previous work, in which we used fluorescent antibodies to detect and quantify protein levels by single molecule counting, demonstrates that this approach is realistic and powerful.

Public Health Relevance

Most cellular functions are performed by proteins, yet current methods are unable to accurately quantify protein levels and post-translational state in a comprehensive manner. This shortcoming is preventing a quantitative understanding of normal cellular processes, the mechanisms by which they fail, and how these failures lead to disease. To redress this deficiency, we propose to apply recent advances in single-molecule imaging to the field of protein detection. By sequencing single peptide molecules in parallel we will develop a protein analysis tool with unprecedented sensitivity, dynamic range, and utility.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA151197-03
Application #
8321082
Study Section
Special Emphasis Panel (ZCA1-SRLB-Q (M2))
Program Officer
Knowlton, John R
Project Start
2010-09-01
Project End
2014-08-31
Budget Start
2012-09-07
Budget End
2014-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$192,409
Indirect Cost
$65,824
Name
Washington University
Department
Genetics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Tessler, Lee A; Donahoe, Casey D; Garcia, Daniel J et al. (2011) Nanogel surface coatings for improved single-molecule imaging substrates. J R Soc Interface 8:1400-8
Tessler, Lee A; Mitra, Robi D (2011) Sensitive single-molecule protein quantification and protein complex detection in a microarray format. Proteomics 11:4731-5