The overall goal of our project is to develop advanced technologies based on electrospray ionization with mass spectrometry (MS) and ion mobility spectrometry (IMS) for proteomics and structural biology. The structural determination of protein complexes can play an important role in the fundamental understanding of biochemical pathways. New techniques will be developed to facilitate the characterization of noncovalent protein assemblies. Information related to protein size, protein folding, and protein conformation that can be used to complement information derived from more traditional high resolution structural methods (e.g., x-ray crystallography, nuclear magnetic resonance, cryoelectron microscopy) will be derived from study of the gas phase protein complexes. Modifications to ion mobility spectrometry systems will allow temperature dependences of the ions traveling in the IMS to be used to follow protein folding/unfolding. Experimental and instrumental methods to reduce the charge states of the gas phase complexes, such as gas phase proton transfer reactions, will be developed;cross-sections from their mobility profiles will be determined to study the structural affects of assembly and ligand binding (i.e., conformational differences). Novel developments in intact protein tandem MS will be exploited to define the sites of ligand and protein interactions in a given noncovalent protein complex. Top-down MS/MS will be developed further as a means to determine the specific sites of small molecule ligand binding. The ability to increase the multiple charging of large noncovalent gas phase molecules will be implemented to increase the efficiency of the top-down MS analysis of protein-ligand complexes. The developed methods will be used to determine the binding sites of metals to metalloproteins involved in neurodegenerative disorders and ATP to protein kinases. MS/MS will be used to deliver a "signature" for ATP-binding that can be used to identify new protein kinases. ESI-MS and MS/MS of protein-ligand binding from structurally diverse compound libraries will be developed as a complementary tool for fragment-based ligand design strategies.

Public Health Relevance

We aim to develop more robust analytical technologies that can be applied to provide structural information for biologically important proteins, such as proteins involved in neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease). New methodologies to determine to sites of drug binding to targeted proteins will be developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
8R01GM103479-09
Application #
8298642
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Sheeley, Douglas
Project Start
2004-07-15
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
9
Fiscal Year
2012
Total Cost
$378,793
Indirect Cost
$131,293
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Kumar, Yogesh; Vadivel, Kanagasabai; Schmidt, Amy E et al. (2014) Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain. Biochemistry 53:505-17
Lakshmanan, Rajeswari; Wolff, Jeremy J; Alvarado, Rudy et al. (2014) Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods. Proteomics 14:1271-82
Ogorzalek Loo, Rachel R; Lakshmanan, Rajeswari; Loo, Joseph A (2014) What protein charging (and supercharging) reveal about the mechanism of electrospray ionization. J Am Soc Mass Spectrom 25:1675-93
Nguyen, Thi H; Kim, Sung-Hye; Decker, Caitlin G et al. (2013) A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor. Nat Chem 5:221-7
Loo, Rachel R Ogorzalek; Loo, Joseph A (2013) Protein complexes: breaking up is hard to do well. Structure 21:1265-6

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