Event Information
Development of Analytical Strategies to Facilitate Protein-Ligand Binding Analysis on the Proteomic Scale
- Abstract:
Currently, the SPROX (Stability of Proteins from Rates of Oxidation) technique is useful for detecting and quantifying protein-ligand binding interactions by selectively oxidizing methionine residues in proteins. The SPROX technique has also been used with quantitative mass spectrometry-based proteomic analysis in conjunction with isobaric mass tags. To date, approximately 500 peptides from approximately 350 proteins have been identified in a typical SPROX experiment with 1-D liquid chromatography–tandem mass spectrometry (LC-MS/MS) and a methionine-containing-peptide enhancement strategy. The strategies presented here, including gel electrophoresis fractionation and gas-phase fractionation of derivatized methionine-containing peptides, aim to increase proteomic coverage and selectivity for the detection and identification of methionine-containing peptides, as well as to improve quantitation capabilities for studies in protein-ligand interactions. Results obtained, to date, have shown that gel electrophoresis can help increase the proteome coverage while identifying complementary peptides to the current solution-based SPROX analysis. As for the gas-phase fractionation, it has been successful; 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 to improve proteomic coverage and methionine selectivity as well as maintain quantitation capabilities for studies in protein-ligand interactions, and thus can help us better study thermodynamic stability of proteins and protein-ligand interactions on proteomic scale.
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