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<p>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.</p><p>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.</p><p>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. </p><p>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.</p>
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