Radiolabeling for two-dimensional gel analysis.

Radiolabeling is a highly sensitive method for protein detection, which is easily performed by the incorporation of radioactive amino acids into proteins. This makes radiolabeling a method of choice for visualizing proteins separated on two-dimensional (2-D) gels. This chapter presents protocols to determine in vivo labeling conditions and to label proteins for the comparison of protein samples by means of 2-D gel electrophoresis.

Two-dimensional gel electrophoresis of total yeast proteins.

Two-dimensional gel electrophoresis (2-DE) offers the opportunity of separating several hundred proteins from a total yeast cellular extract. A detailed description is provided here of the different steps required for the separation and visualization of radiolabeled yeast proteins on high-resolution (24 cm x 20 cm) 2-D gels. Two methods of protein separation are described. They essentially differ by the way proteins are separated in the first dimension. One is based on the use of isoelectric focusing (IEF) gels (carrier ampholytes) and the other on the use of ready-made IPG gels (immobilines). These methods allow separating soluble proteins from a total yeast cellular extract with an isoelectric point ranging between pH 4.0 and 7.0 and a molecular weight ranging between 15,000 and 150,000.

Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples.

We describe a novel methodology for the comparison of protein samples called differential gel exposure (DifExpo). This method is based on the coelectrophoresis on a two-dimensional (2-D) gel of two protein samples. The samples are differentiated from each other by in vivo radiolabelling, using (14)C- and (3)H-isotopes. After 2-D separation and transfer on a polyvinylidene difluoride membrane, the (3)H/(14)C ratio of each protein spot is determined by exposure to two types of imaging plates, one sensitive to (14)C and the other to both (14)C and (3)H. We showed that DifExpo allows us to compare the cellular levels of several hundred proteins of the yeast proteome. Its sensitivity is comparable to silver staining. We also showed that it can be used to investigate changes in the rate of synthesis of individual proteins.

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.