High-throughput chemical analysis has become critical to progress in biomedical research. High-throughput is used in drug discovery, drug development, characterizing combinatorial synthetic reactions, diagnostics, and new biotechnologies such as directed evolution of proteins. Presently high-throughput chemical analysis is dominated by fluorescence assays performed on multi-well plates. This approach has important limitations including false positives and the cost and time of building fluorescence changes into biochemical reactions. Mass spectrometry (MS) and electrophoresis are highly promising as high-throughput analysis methods that can be performed with minimal or no requirements for labels by directly detecting biochemicals involved in reactions. They also offer potential for multiplexing by resolving multiple compounds in one assay. Some progress has been made in developing these methods for high-throughput analysis;however, current approaches do not offer throughput comparable to fluorescence. We propose to couple segmented flow, where samples are compartmentalized as nanoliter droplets within an immiscible fluid, directly to MS and electrophoresis. This approach will allow analysis rates up to 10 Hz while consuming only nanoliters of sample. The latter is important because of the reduction in cost associated with screening. Because rapid analysis cannot be achieved without high-throughput sample preparation, we also propose to develop novel approaches to preparing samples for MS and electrophoresis including high-throughput extraction. These new methods will be applied to screening for modulators of novel protein targets that presently do not have high-throughput assays available. We will target Hsp70-protein complex formation, sirtuin 5 desuccinylase, sirtuin 6 deacetylase, and cytochrome P450 CYP2E1 activities. These proteins are implicated in Alzheimer's disease, type 2 diabetes, cancer, and alcohol liver disease, respectively. The significance of this work is that it provides powerful new technology for high-throughput chemical analysis on a highly miniaturized scale to increase the pace of drug development and reduce costs. It also will identify chemical leads for several novel protein targets. Important innovations include segmented flow sample introduction for MS and electrophoresis, high-throughput sample preparation, and development of assays for novel protein targets.

Public Health Relevance

High-throughput chemical analysis has become increasingly important in biomedical research. High throughput is used in drug discovery, drug development, diagnostics, and biotechnology. This work will develop improved methods of high-throughput chemical analysis that may be used in such applications. The newly developed methods will be used in screens for modulators of proteins involved in type 2 diabetes, Alzheimer's disease, cancer, and alcohol liver disease. Lead compounds developed from these screens may prove useful in treatments for these diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM102236-02
Application #
8545872
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2012-09-30
Project End
2016-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
2
Fiscal Year
2013
Total Cost
$272,010
Indirect Cost
$88,660
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Guetschow, Erik D; Steyer, Daniel J; Kennedy, Robert T (2014) Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators. Anal Chem 86:10373-9
Sun, Shuwen; Kennedy, Robert T (2014) Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors. Anal Chem 86:9309-14
Rauch, Jennifer N; Nie, Jing; Buchholz, Tonia J et al. (2013) Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions. Anal Chem 85:9824-31
Nie, Jing; Kennedy, Robert T (2013) Capillary liquid chromatography fraction collection and postcolumn reaction using segmented flow microfluidics. J Sep Sci 36:3471-7
Jin, Shi; Anderson, Gwendolyn J; Kennedy, Robert T (2013) Western blotting using microchip electrophoresis interfaced to a protein capture membrane. Anal Chem 85:6073-9