Measurement of Protein Sulfhydryls in Response to Cellular Oxidative Stress Using Gel Electrophoresis and Multiplexed Fluorescent Imaging Analysis

Abstract

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.

Protein Thiol Oxidation in Murine Airway Epithelial Cells in Response to Naphthalene or Diethyl Maleate

“…The method used in the current work is the same as in a recently published paper describing the procedures for monitoring protein thiol status using differential fluorescence labeling coupled with protein separation by two-dimensional (2D) gel electrophoresis (16). Therefore, abbreviated descriptions are provided here.…”

“…We have recently reported a method that allows measurement of the ratio of reduced to oxidized cellular protein thiols using differential fluorescence labeling combined with gel electrophoresis (16). The current study uses this method to address the differences in protein thiol oxidation caused by NA and DEM in airway epithelial cells of male mice after intraperitoneal administration.…”

“…The airways were then lavaged with lysis buffer containing detergents, protein denaturants, and protease inhibitor cocktail as described (16) to recover airway epithelial proteins. The residual lung, a small liver sample, and 50 ml of lysis lavage sample from each animal were homogenized in Solution A (200 mM methane sulfonic acid, 5 mM diethylenepentaacetic acid) for GSH analysis.…”

“…This method was first described by Spiess and colleagues (16). Each lavage sample, containing approximately 1 mg protein, was added immediately to tubes containing 250 nmol Cy3 maleimide dye for labeling of reduced thiols within the sample.…”

“…Two narrow-range, 18-cm Immobiline DryStrips (GE Health Care, Piscataway, NJ) (pH 4.5-5.5 and pH 5.5-6.7) were rehydrated for each sample. Protein separation by 2D gel electrophoresis was performed as described previously (16). After electrophoresis, the protein gels were fixed in 25% methanol, 5% acetic acid overnight.…”