Skip to main content
Duke University Libraries
DukeSpace Scholarship by Duke Authors
  • Login
  • Ask
  • Menu
  • Login
  • Ask a Librarian
  • Search & Find
  • Using the Library
  • Research Support
  • Course Support
  • Libraries
  • About
View Item 
  •   DukeSpace
  • Theses and Dissertations
  • Duke Dissertations
  • View Item
  •   DukeSpace
  • Theses and Dissertations
  • Duke Dissertations
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

Application and Evaluation of a Chemical Modification- and Mass Spectrometry-Based Thermodynamic Assay for the Study of Protein-Ligand Interactions in Complex Mixtures

Thumbnail
View / Download
3.0 Mb
Date
2013
Author
Strickland, Erin Catherine
Advisor
Fitzgerald, Michael C
Repository Usage Stats
465
views
380
downloads
Abstract

While a number of different proteomic, genomic, and computational approaches exist for the characterization of drug action, each of the experimental approaches developed to date has both strengths and weaknesses. Currently, there is no one "perfect" assay for drug mode-of-action studies. A protocol that could assay all the proteins in the proteome for both direct and indirect binding interactions of drugs would greatly facilitate studies of drug action. Recently, the SPROX (stability of proteins from rates of oxidation) technique was developed as a chemical modification- and mass spectrometry-based strategy for detecting protein-ligand interactions by monitoring the change in thermodynamic stability of proteins upon ligand binding. This is accomplished by monitoring the denaturant dependent oxidation of globally protected methionine residues. The SPROX technique has been interfaced with bottom-up proteomics methods to allow for the proteome-wide analysis of protein-ligand interactions. However, the strategy has been limited by the need to detect and quantify methionine containing peptides in the bottom-up proteomics experiment.

The work in this dissertation is focused on evaluating the current SPROX protocol, developing modifications to improve proteome coverage, and applying the SPROX platform to two different drug mode-of-action studies. Three main strategies were employed to improve protein coverage. First, a chemo-selective isolation of un-oxidized methionine containing peptides was employed to enrich for methionine containing peptides, and it was found to produce a ~2-fold improvement in proteomic coverage. Second, a pre-fractionation strategy involving the use of isoelectric focusing was employed to decrease sample complexity prior to LC-MS/MS analysis and it was found to generate a ~2-3 fold improvement in proteomic coverage, however when combined with the methionine enrichment strategy the improvement was ~6-fold as the benefits of both were additive. Third, a tryptophan modification strategy was developed that could ultimately expand the number of useful peptides in proteome-wide SPROX experiments to include those that contain tryptophan. Also, investigated was the use of several different mass spectrometer systems (including a bench-top quadrupole and orbitrap system and two different quadrupole time-of-flight systems) in the SPROX protocol. The results of these studies indicate that there is a significant advantage in proteome coverage when faster mass spectrometers are used. The use of high energy collision dissociation (HCD) in the orbitrap system was also more advantageous than the use of collision induced dissociation (CID) in the Q-ToF systems. Regardless of the mass spectrometer used, the major source of error in the SPROX experiment was found to be the random error associated with the LC-MS/MS analysis of isobaric mass tagged peptides. This random error was found to yield a false discovery rate of between 3 and 10% for "hit" peptides in the SPROX experiment.

The above improvements in the SPROX protocol were used in two protein-ligand binding experiments. One set of experiments involved studies on two small molecules with a specific anti-cancer phenotype in human colon cancer cells. These studies identified 17 proteins as potential "hits" of these two small molecules. After preliminary validation of these proteins, approximately 50% were eliminated as false positives and one protein, p80/nucleophosim, showed consistent data indicating a destabilizing interaction with both small molecules. The destabilization is indicative of an indirect interaction with the small molecules that would be mediated through a protein-protein interaction network. In another set of experiments the breast cancer drug, tamoxifen, and its main, active metabolite, 4-hydroxy tamoxifen, were assayed for binding to the proteins in a yeast cell lysate to better understand its adverse effects on yeast cells. The results of these studies identified ~80 proteins as potential "hits" of these two drugs. After preliminary validation of these proteins, approximately 30% were eliminated as false positives and one protein, SIS1, type II Hsp40, showed consistent data indicative of a direct binding interaction.

Type
Dissertation
Department
Chemistry
Subject
Chemistry
drug mode-of-action
protein-ligand binding
protein thermodynamic stability
SPROX
Permalink
https://hdl.handle.net/10161/7241
Citation
Strickland, Erin Catherine (2013). Application and Evaluation of a Chemical Modification- and Mass Spectrometry-Based Thermodynamic Assay for the Study of Protein-Ligand Interactions in Complex Mixtures. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/7241.
Collections
  • Duke Dissertations
More Info
Show full item record
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 United States License.

Rights for Collection: Duke Dissertations


Works are deposited here by their authors, and represent their research and opinions, not that of Duke University. Some materials and descriptions may include offensive content. More info

Make Your Work Available Here

How to Deposit

Browse

All of DukeSpaceCommunities & CollectionsAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit DateThis CollectionAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit Date

My Account

LoginRegister

Statistics

View Usage Statistics
Duke University Libraries

Contact Us

411 Chapel Drive
Durham, NC 27708
(919) 660-5870
Perkins Library Service Desk

Digital Repositories at Duke

  • Report a problem with the repositories
  • About digital repositories at Duke
  • Accessibility Policy
  • Deaccession and DMCA Takedown Policy

TwitterFacebookYouTubeFlickrInstagramBlogs

Sign Up for Our Newsletter
  • Re-use & Attribution / Privacy
  • Harmful Language Statement
  • Support the Libraries
Duke University