The long-term goal of this project is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Immobilize low concentrations of different classes of enzymes with high efficiency and with retention of enzyme activity for the purpose of screening large numbers of inhibitor libraries (ligands) using our newly developed IEMS method. 2) Identify possible inhibitors showing at least 40 percent inhibition and apply MS and MS/MS as needed to ensure correct combinatorial synthesis and identify synthetic byproducts, 3) Calculate Km, Vmax, and kcat, as needed, of both immobilized and soluble enzymes using our MS method. 4) Determine if inhibition is competitive or non-competitive. 5) Calculate Ki's for inhibitors. It is anticipated that large numbers of compounds can be screened using our IEMS method with either ion trap- or FTICR-MS without the need of chromatography. The high resolution and high mass accuracy of the FTICR will allow for separation of isobars in the IEMS assay. Multiple stages of MS will provide structural identification of both desired synthetic products of the combinatorial synthesis and of unanticipated synthetic byproducts. It is additionally proposed that kinetic parameters can be calculated using ESI-ion trap mass spectrometry thus obviating the need for chromophores as required by traditional spectrophotometric techniques. A collaboration has been established with Professor Carolyn Bertozzi in which projects involving estrogen sulfotransferase (EST) and glycosyl sulfotransferase (NoST) will be investigated in addition to other enzymatic systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063581-05
Application #
6857144
Study Section
Special Emphasis Panel (ZRG1-BECM (01))
Program Officer
Edmonds, Charles G
Project Start
2002-03-01
Project End
2006-12-14
Budget Start
2005-03-01
Budget End
2006-12-14
Support Year
5
Fiscal Year
2005
Total Cost
$195,520
Indirect Cost
Name
University of California Davis
Department
Genetics
Type
Schools of Arts and Sciences
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Jen, Connie H; Leary, Julie A (2010) A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry. Anal Biochem 407:134-40
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Jia, Weitao; Shaffer, Justin F; Harris, Samantha P et al. (2010) Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis. J Proteome Res 9:1843-53
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Gao, Hong; Leary, Julie; Carroll, Kate S et al. (2007) Noncovalent complexes of APS reductase from M. tuberculosis: delineating a mechanistic model using ESI-FTICR MS. J Am Soc Mass Spectrom 18:167-78
Yu, Yonghao; Hoffhines, Adam J; Moore, Kevin L et al. (2007) Determination of the sites of tyrosine O-sulfation in peptides and proteins. Nat Methods 4:583-8
Yu, Yonghao; Sweeney, Matthew D; Saad, Ola M et al. (2006) Potential inhibitors of chemokine function: analysis of noncovalent complexes of CC chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry. J Am Soc Mass Spectrom 17:524-35
Crown, Susan E; Yu, Yonghao; Sweeney, Matthew D et al. (2006) Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding. J Biol Chem 281:25438-46

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