| NSF Org: |
CHE Division Of Chemistry |
| Recipient: |
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| Initial Amendment Date: | August 30, 2013 |
| Latest Amendment Date: | August 30, 2013 |
| Award Number: | 1308093 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Kelsey Cook
CHE Division Of Chemistry MPS Directorate for Mathematical and Physical Sciences |
| Start Date: | September 15, 2013 |
| End Date: | December 31, 2016 (Estimated) |
| Total Intended Award Amount: | $407,000.00 |
| Total Awarded Amount to Date: | $407,000.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
2200 W MAIN ST DURHAM NC US 27705-4640 (919)684-3030 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
NC US 27705-4010 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Chemical Measurement & Imaging |
| Primary Program Source: |
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| Program Reference Code(s): | |
| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.049 |
ABSTRACT
With support form the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry, Professor Michael Fitzgerald and his research group at Duke University will develop and apply new quantitative mass spectrometry-based proteomics methods for the large-scale thermodynamic analysis of protein-ligand binding interactions. The specific protocols under development utilize the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with a stable isotope labeling with amino acids in cell culture (SILAC) approach to compare the equilibrium unfolding/folding properties of proteins and protein ligand-complexes in real biological mixtures (e.g., cell lysates). The new protocols, which are designed to significantly expand the number of proteins assayed in proteome-wide SPROX experiments, will be benchmarked in several proof-of-principle experiments in which the endogenous proteins in a yeast cell lysate are analyzed for binding to ATP (a ubiquitous enzyme cofactor), to geldanamycin (an Hsp90 inhibitor), and to cyclosporine A (an immunosuppressive agent that directly binds cyclophilin A and indirectly interacts with calcineurin. The new protocols will also be used in a real drug mode-of-action studies involving manassantin A, which is a natural product with demonstrated anti-cancer activity in cell-based assays but an as yet unknown mode-of-action.
The strategies and protocols to be created will be readily transferable to almost any protein-ligand system or biological mixture regardless of the ligand (i.e. small molecule, DNA, peptide, or other proteins) or binding affinity. The research provides interdisciplinary research experiences for both graduate and undergraduate students, and as well as high school students from the North Carolina Project SEED program, which is an on-going science enrichment program designed to steer disadvantaged, underrepresented minority students from North Carolina high schools into chemistry and chemistry-related careers.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
The research conducted under this award resulted in the development of four new protein mass spectrometry methods designed to make proteome-wide measurements of protein folding stabilities. Of particular significance to the overall goals of the work was that the newly developed methods significantly expanded the scope of the Stability of Proteins from Rates of Oxidation (SPROX) methodology, previously developed by the PI. The SPROX-related methods developed here enabled the protein folding properties of on the order of a 1,000 proteins from complex biological mixtures such as cell lysates to be measured in a single experiment. The utility of this new capability was demonstrated in a protein target discovery project involving manassantin A, which is a natural product that has known anti-cancer activities but a poorly understood mode of action. The developed methods were also used to uncover previously unknown biophysical properties of protein allergens in the dust mite proteome. The dust mite protein allergens were generally found to be among the more abundant and more stable proteins in the proteome.
The work performed under this award also contributed to the academic training of 6 graduate students at Duke University, 4 of whom were female. A female high school student researcher also worked on the project during the summers of 2015 and 2016 through the American Chemical Society’s Project SEED Program; and the data she generated was included in a science project that she submitted to several North Carolina statewide science fair competitions, to the Siemans Competition in Math, Science, and Technology and to the Intel International Science and Engineering Fair. The research experience the award help provide to this high school student bolstered her college applications, and she is currently on track to be the first in her family to attend college.
The research results generated under this award have also been included in 8 research publications including 2 in the Journal of Analytical Chemistry, 3 in the Journal of the American Society for Mass Spectrometry, 1 in Molecular & Cellular Proteomics, 1 in the Journal of Proteome Research, and 1 in the Journal of Allergy and Clinical Immunology. The PI and one of the graduate student researchers working on this project have also co-authored a book chapter in Annual Review of Analytical Chemistry on the general subject of energetics-based methods for protein folding and stability measurements.
Last Modified: 03/29/2017
Modified by: Michael C Fitzgerald
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