Evaluation of Energetics-based Techniques for Proteome-Wide Studies of Protein-Ligand Binding Interactions

dc.contributor.advisor

Fitzgerald, Michael C

dc.contributor.author

Geer, Michelle Ariel

dc.date.accessioned

2015-05-12T20:45:37Z

dc.date.available

2017-04-27T04:30:03Z

dc.date.issued

2015

dc.department

Chemistry

dc.description.abstract

Detection and quantification of protein-ligand binding interactions is extremely important for understanding interactions that occur in biological systems. Since traditional techniques for characterizing these types of interactions cannot be performed in complex systems such as cell lysates, a series of energetics-based techniques that are capable of assessing protein stability and measuring ligand binding affinities have been developed to overcome some of the limitations of previous techniques. Now that the capabilities of the energetics-based techniques have been exhibited in model systems, the false-positive rates of the techniques, the range of biological questions to which the techniques can be addressed, and the use of the techniques to discover novel interactions in unknown systems remained to be shown. The Stability of Proteins from Rates of Oxidation (SPROX) technique and the Pulse Proteolysis (PP) technique were applied to a wide range of biological questions in both yeast and human cell lysates to evaluate the scope of these experimental workflows. The false-positive rate of iTRAQ-SPROX protein target discovery on orbitrap mass spectrometer systems was determined to be < 0.8 %. The iTRAQ-SPROX technique was successfully applied to the discovery of both known and novel protein-protein, protein-ATP, and protein-drug interactions, leading to the quantification of protein-ligand binding affinities in each of these studies. In the pursuit of discovering geldanamycin protein interactors, the use of iTRAQ-SPROX and SILAC-PP in combination was determined to be advantageous for confirming protein-ligand interactions since the techniques utilize different quantitation strategies that are subject to separate technical errors in quantitation. Finally, the iTRAQ-SPROX and SILAC-PP techniques were used to evaluate the interactions of manassantin A in a human cell lysate. In this work, a previously unknown protein target of manassantin A, Filamin A, was detected as a hit protein using both the iTRAQ-SPROX and SILAC-PP protocols. The work completed in this dissertation has expanded the understanding of the limitations of energetics-based techniques and shown that biological replicate analyses are essential to confirm ligand interactions with novel protein targets.

dc.identifier.uri

https://hdl.handle.net/10161/9905

dc.subject

Analytical chemistry

dc.subject

Biochemistry

dc.subject

mass spectrometry

dc.subject

protein-ligand interactions

dc.subject

protein stability

dc.subject

pulse proteolysis

dc.subject

stability of proteins from rates of oxidation

dc.subject

thermodynamics

dc.title

Evaluation of Energetics-based Techniques for Proteome-Wide Studies of Protein-Ligand Binding Interactions

dc.type

Dissertation

duke.embargo.months

23

Files

Original bundle

Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Geer_duke_0066D_12866.pdf
Size:
7.41 MB
Format:
Adobe Portable Document Format

Collections