| dc.description.abstract |
<p>SPROX (Stability of Proteins from Rates of Oxidation) is a technique for the detection
and quantitation of protein-ligand binding interactions. In SPROX the differential
denaturant dependence of a methionine oxidation reaction in proteins performed in
the presence and in the absence of ligand is used to measure the thermodynamic properties
of protein-ligand binding interactions. Recently, the SPROX technique has also been
used with quantitative mass spectrometry-based proteomic strategies to simultaneously
assay up to hundreds of different proteins in complex biological samples. The proteomic
applications of SPROX, to date, have relied on the use of a methionine-containing-peptide
enhancement strategy and 1-D liquid chromatography-tandem mass spectrometry (LC-MS/MS)
and. The strategies presented here, including gel electrophoresis fractionation and
gas-phase fractionation of derivatized methionine-containing peptides, aim to increase
proteomic coverage in SPROX by facilitating the detection, identification, and quantitation
of methionine-containing peptides in the proteomics experiment. The initial results
obtained in this work have shown that gel electrophoresis can help increase the proteome
coverage while identifying complementary peptides to the current solution-based SPROX
analysis. A gas-phase fractionation strategy is also demonstrated; however, the mass
spectrometric analysis needs to be optimized and further tested to achieve selectivity
for methionine peptides. Both of these fractionation strategies show promise for improving
the proteomic coverage in SPROX studies of protein-ligand interactions on the proteomic
scale. In addition, the previously established SPROX protocol using isobaric mass
tags was used in two protein-ligand binding experiments, including one experiment
to identify the protein targets in a yeast cell lysate of adenosine 5'-(β,γ-imido)triphosphate
(AMP-PNP), a non-hydrolyzable adenosine-5'-triphosphate (ATP) mimic, and second experiment
to investigate the protein targets of two iron chelators, deferasirox (Exjade) and
N'-[1-(2hydroxyphenyl) ethyliden]isonicotinoylhydrazide (HAPI), both of which protect
ARPE-19 cells from oxidative damage induced by hydrogen peroxide. Three known ATP-binding
proteins, ADEnine requiring, URAcil requiring and Yeast Elongation Factor, have been
identified, while further investigation for the protein targets of iron chelators
needs to be performed.</p>
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