Dynamics of protein damage in yeast frataxin mutant exposed to oxidative stress.
Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.
Kim
Jin-Hee
JH
Bindley Bioscience Center at Discovery Park, Purdue University, West Lafayette, Indiana, USA.
Sedlak
Miroslav
M
Gao
Qiang
Q
Riley
Catherine P
CP
Regnier
Fred E
FE
Adamec
Jiri
J
D017353:Gene Deletion; D006861:Hydrogen Peroxide / Q000494:pharmacology; D033862:Iron-Binding Proteins / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050050:Protein Carbonylation / Q000187:drug effects / Q000235:genetics; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000096923:Frataxin
D006861:Hydrogen Peroxide; D033862:Iron-Binding Proteins; D010544:Peroxidases; D029701:Saccharomyces cerevisiae Proteins; D013879:Thioredoxins; D000096923:Frataxin; C516110:TRX2 protein, S cerevisiae; C496253:Tsa1 protein, S cerevisiae