Detection and Quantification of Protein Disulfides in Biological Tissues: A Fluorescence-Based Proteomic Approach

Viviana I. Pérez, Anson Pierce, Eric M. de Waal, Walter F. Ward, Alex Bokov, Asish Chaudhuri, Arlan Richardson

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

Abstract

While most of the amino acids in proteins are potential targets for oxidation, the thiol group in cysteine is one of the most reactive amino acid side chains. The thiol group can be oxidized to several states, including the disulfide bond. Despite the known sensitivity of cysteine to oxidation and the physiological importance of the thiol group to protein structure and function, little information is available on the oxidative modification of cysteine residues in proteins because of the lack of reproducible and sensitive assays to measure cysteine oxidation in the proteome. We have developed a fluorescence-based assay that allows one to quantify both the global level of protein disulfides in the cellular proteome as well as the disulfide content of individual proteins. This fluorescence-based assay is able to detect an increase in global protein disulfide levels after oxidative stress in vitro or in vivo. Using this assay, we show that the global protein disulfide levels increase significantly with age in liver cytosolic proteins, and we identified 11 proteins that show a more than twofold increase in disulfide content with age. Thus, the fluorescence-based assay we have developed allows one to quantify changes in the oxidation of cysteine residues to disulfides in the proteome of a cell or tissue.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages161-177
Number of pages17
DOIs
StatePublished - Jan 2010

Publication series

NameMethods in Enzymology
Volume473
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Aging
  • Cysteine oxidation
  • Protein disulfide
  • Protein oxidation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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