Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach.

Published

Journal Article

Shotgun proteomics protocols are widely used for the identification and/or quantitation of proteins in complex biological samples. Described here is a shotgun proteomics protocol that can be used to identify the protein targets of biologically relevant ligands in complex protein mixtures. The protocol combines a quantitative proteomics platform with a covalent modification strategy, termed Stability of Proteins from Rates of Oxidation (SPROX), which utilizes the denaturant dependence of hydrogen peroxide-mediated oxidation of methionine side chains in proteins to assess the thermodynamic properties of proteins and protein-ligand complexes. The quantitative proteomics platform involves the use of isobaric mass tags and a methionine-containing peptide enhancement strategy. The protocol is evaluated in a ligand binding experiment designed to identify the proteins in a yeast cell lysate that bind the well-known enzyme cofactor, β-nicotinamide adenine dinucleotide (NAD+). The protocol is also used to investigate the protein targets of resveratrol, a biologically active ligand with less well-understood protein targets. A known protein target of resveratrol, cytosolic aldehyde dehydrogenase, was identified in addition to six other potential new proteins targets including four that are associated with the protein translation machinery, which has previously been implicated as a target of resveratrol.

Full Text

Duke Authors

Cited Authors

  • Dearmond, PD; Xu, Y; Strickland, EC; Daniels, KG; Fitzgerald, MC

Published Date

  • November 2011

Published In

Volume / Issue

  • 10 / 11

Start / End Page

  • 4948 - 4958

PubMed ID

  • 21905665

Pubmed Central ID

  • 21905665

Electronic International Standard Serial Number (EISSN)

  • 1535-3907

International Standard Serial Number (ISSN)

  • 1535-3893

Digital Object Identifier (DOI)

  • 10.1021/pr200403c

Language

  • eng