Fig. 13.3. Oxidized protein-thiols were labeled with [14C]NEM and separated by 2D-gel electrophoresis, as described in Sect. 3.2.2. Exponentially growing wild-type yeast cells were either left untreated or were treated during 1 min with 1 mM H2O2. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Tdh3) and the peroxiredoxin Ahp1 increase in oxidation upon H2O2 treatment.

the radioactive signal of their 2D-gels spot, also providing estimates of protein redox ratios, when accurate controls are used. Fluorescent derivatives of IAM (16), and NEM (17), made by their fusion with fluorescent dyes, require the analysis of 2D-gels by fluorimetry (Fig.13.4). These dyes have the virtue of providing a very accurate measure of the redox ratio of a given substrate, when dyes of different spectral emission are used to differentially label reduced and oxidized cysteine residues respectively. It should be said that these proteome-based thiol-labeling methods are fraught with a relatively high noise background that likely is produced by thiol-nonspecific protein-adduct formation, which warrant the use of the adequate controls to subtract noise from specific signals. Oxidized HPDP is an absolute thiol-specific reagent that covalently and

Protein-Thiols Purification reversibly attaches to free thiol groups through a disulfide bridge

(14, 18). Labeling of oxidized protein-thiols using biotin-HPDP allows their purification by streptavidin affinity (Fig.13.5). Elu-tion of streptavidin-bound proteins involves reduction of the disulfide linkage between them and biotin-HPDP, using DTT. These purified proteins can then be separated by 2D gel elec-trophoresis, and identified by mass spectrometry. This method is only qualitative. Biotin-HPDP-modified proteins can also be recognized by streptavidin western blot. Biotin-NEM and Biotin-IAM exist and can also be used for purification of oxidized protein-thiols, or in streptavidin western blots.

Wild-type - H2O2



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