Development and Application of Analytical Strategies to Facilitate Protein-Ligand Binding Analysis on the Proteomic Scale

Abstract

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.