Genome- and proteome-based gene expression profiling studies have been widely used over the past decade to characterize biological states and drug action. While such differential gene expression profiling studies can help identify the cellular pathways and physiological processes associated with a biological state or with the biological activity of a therapeutic agent, they often fail to produce a detailed molecular level understanding of the biological state or drug mode-of-action because gene expression levels are not directly tied to protein function. This has limited the number of useful protein biomarkers and therapeutic targets discovered from such studies, and left gaps in our understanding of drug-action. Proposed here is the use of thermodynamic measurements of protein stability to profile the protein-interaction networks associated with different biological states and drug action. Such thermodynamic measurements of protein stability are expected to be more closely related to protein function than are protein expression levels, and thus produce more useful protein biomarkers and therapeutic targets of disease and generate a better understanding of drug action. The proposed work will use a chemical modification- and mass spectrometry-based method, termed SPROX, to make thermodynamic measurements of protein stability on the proteomic scale in several different age-, disease-, and drug-related biological states.
The specific aims of this work are: (1) to streamline and improve the data analysis pipeline in proteome-wide SPROX experiments;(2) to characterize the thermodynamic stability of proteins derived from four different cell culture models of breast cancer using the SPROX technique and determine which proteins have altered stabilities in the different cell culture models;(3) to characterize the thermodynamic stability of proteins derived from breast cancer cells grown in the presence and in the absence of tamoxifen using the SPROX technique and determine which proteins have altered stabilities in the presence of tamoxifen;(4) to characterize the thermodynamic stability of mouse proteins derived from a mouse model of aging using the SPROX technique and determine which proteins have altered stabilities as a function of age;and (5) to use the proteins identified in (2)-(4) to characterize the altered protein interaction networks associated with the different age-, disease-, and drug-related biological states in this study.

Public Health Relevance

Currently, there is much effort focused on using genome- and proteome-based gene expression profiling studies to diagnose and understand the biological processes associated with aging, disease, and drug action. Proposed here is an effort to investigate the use of thermodynamic measurements of protein stability for characterizing the biological states such as those associated with aging, disease, and drug action. Such thermodynamic measurements of protein stability are hypothesized to be more closely related to protein function than are protein expression levels, and thus expected to produce more useful protein biomarkers and therapeutic targets of disease and to generate a better understanding of drug action and disease biology.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM084174-05A1
Application #
8692475
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Duke University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Durham
State
NC
Country
United States
Zip Code
27705
Strickland, Erin C; Geer, M Ariel; Hong, Jiyong et al. (2014) False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform. J Am Soc Mass Spectrom 25:132-40
Tran, Duc T; Adhikari, Jagat; Fitzgerald, Michael C (2014) StableIsotope Labeling with Amino Acids in Cell Culture (SILAC)-based strategy for proteome-wide thermodynamic analysis of protein-ligand binding interactions. Mol Cell Proteomics 13:1800-13
Strickland, Erin C; Geer, M Ariel; Tran, Duc T et al. (2013) Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation. Nat Protoc 8:148-61
Reichmann, Dana; Xu, Ying; Cremers, Claudia M et al. (2012) Order out of disorder: working cycle of an intrinsically unfolded chaperone. Cell 148:947-57
Banerjee, Sambuddha; Siburt, Claire J Parker; Mistry, Shreni et al. (2012) Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport. Metallomics 4:361-72
Tran, Duc T; Banerjee, Sambuddha; Alayash, Abdu I et al. (2012) Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry. Anal Chem 84:1653-60
Dearmond, Patrick D; Xu, Ying; Strickland, Erin C et al. (2011) Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach. J Proteome Res 10:4948-58
DeArmond, Patrick D; West, Graham M; Huang, Hai-Tsang et al. (2011) Stable isotope labeling strategy for protein-ligand binding analysis in multi-component protein mixtures. J Am Soc Mass Spectrom 22:418-30
Xu, Ying; Schmitt, Sebastian; Tang, Liangjie et al. (2010) Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates. Biochemistry 49:1346-53