Detection and quantification of protein disulfides in biological tissues a fluorescence-based proteomic approach.
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.
Department of Cellular & Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Viviana I
VI
Pérez
Anson
A
Pierce
Eric M
EM
de Waal
Walter F
WF
Ward
Alex
A
Bokov
Asish
A
Chaudhuri
Arlan
A
Richardson
D000375:Aging / Q000378:metabolism / Q000502:physiology; D000825:Animal Structures / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D004220:Disulfides / Q000032:analysis* / Q000378:metabolism; D005453:Fluorescence; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018384:Oxidative Stress / Q000502:physiology; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D004220:Disulfides; D011506:Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)