A novel strategy for global analysis of the dynamic thiol redox proteome.
Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.
Centro de Biología Molecular Severo Ochoa, Nicolás Cabrera 1, 28049 Madrid, Spain.
Pablo
P
Martínez-Acedo
Estefanía
E
Núñez
Francisco J Sánchez
FJ
Gómez
Margoth
M
Moreno
Elena
E
Ramos
Alicia
A
Izquierdo-Álvarez
Elisabet
E
Miró-Casas
Raquel
R
Mesa
Patricia
P
Rodriguez
Antonio
A
Martínez-Ruiz
David Garcia
DG
Dorado
Santiago
S
Lamas
Jesús
J
Vázquez
D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011506:Proteins / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D026361:Reactive Nitrogen Species / Q000032:analysis / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*
D011506:Proteins; D020543:Proteome; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds