Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding

dc.contributor.advisor

Fitzgerald, Michael C

dc.contributor.author

Hopper, Erin D.

dc.date.accessioned

2009-05-01T18:26:38Z

dc.date.available

2011-07-26T04:30:04Z

dc.date.issued

2009

dc.department

Chemistry

dc.description.abstract

Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.

This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.

The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.

To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.

dc.format.extent

2142476 bytes

dc.format.mimetype

application/pdf

dc.identifier.uri

https://hdl.handle.net/10161/1117

dc.language.iso

en_US

dc.subject

Chemistry, Analytical

dc.subject

Biophysics, General

dc.subject

Chemistry, Biochemistry

dc.subject

H

dc.subject

D exchange

dc.subject

high throughput screening

dc.subject

MALDI

dc.subject

mass spectrometry

dc.subject

protein

dc.subject

ligand binding

dc.subject

thermodynamics

dc.title

Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding

dc.type

Dissertation

duke.embargo.months

24

Files

Original bundle

Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
D_Hopper_Erin_a_200904.pdf
Size:
2.04 MB
Format:
Adobe Portable Document Format

Collections