Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.
Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H(2)O(2)) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30-90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.
Leichert
Lars I
LI
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109, USA.
Gehrke
Florian
F
Gudiseva
Harini V
HV
Blackwell
Tom
T
Ilbert
Marianne
M
Walker
Angela K
AK
Strahler
John R
JR
Andrews
Philip C
PC
Jakob
Ursula
U
D000818:Animals; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D020543:Proteome; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism*
D029968:Escherichia coli Proteins; D020543:Proteome; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide