Measurement of protein sulfhydryls in response to cellular oxidative stress using gel electrophoresis and multiplexed fluorescent imaging analysis.
The significance of free radicals in biology has been established by numerous investigations spanning a period of over 40 years. Whereas there are many intracellular targets for these radical species, the importance of cysteine thiol posttranslational modification has received considerable attention. The current studies present a highly sensitive method for measurement of the posttranslational modification of protein thiols. This method is based on labeling of proteins with monofunctional maleimide dyes followed by 2D gel electrophoresis to separate proteins and multiplexed fluorescent imaging analysis. The method correctly interrogates the thiol/disulfide ratio present in commercially available proteins. Exposure of pulmonary airway epithelial cells to high concentrations of menadione or t-butyl hydroperoxide resulted in the modification of cysteines in more than 141 proteins of which 60 were subsequently identified by MALDI-TOF/TOF MS. Although some proteins were modified similarly by these two oxidants, several showed detectably different maleimide ratios in response to these two agents. Proteins that were modified by one or both oxidants include those involved in transcription, protein synthesis and folding, and cell death/growth. In conclusion, these studies provide a novel procedure for measuring the redox status of cysteine thiols on individual proteins with a clearly demonstrated applicability to interactions of chemicals with pulmonary epithelial cells.
Spiess
Page C
PC
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California, USA. pcspiess@ucdavis.edu
Morin
Dexter
D
Jewell
William T
WT
Buckpitt
Alan R
AR
D000818:Animals; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000737:chemistry*; D006868:Hydrolysis; D008297:Male; D008301:Maleimides / Q000737:chemistry; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010720:Phosphines; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D024483:Vitamin K 3 / Q000737:chemistry; D020122:tert-Butylhydroperoxide / Q000737:chemistry
D004220:Disulfides; D005456:Fluorescent Dyes; D008301:Maleimides; D010720:Phosphines; D011506:Proteins; D013438:Sulfhydryl Compounds; C043255:tri-n-butylphosphine; D024483:Vitamin K 3; D020122:tert-Butylhydroperoxide