FIG. 5.

Saturation fluorescence labeling with BODIPY-FL. (A) Schematic diagram of an example of the saturation fluorescence labeling assay for detection of cysteine oxidation. Protein extracts were assayed for cysteine content by amino acid analysis. Enough BODIPY-FL-maleimide was added at pH 7.5 to the unreduced protein mixture to yield a ratio of 60-fold BODIPY to protein thiols. BODIPY will react with reduced cysteine thiols but will not label oxidized thiols. Sypro ruby stain is directed to free amino groups and not affected by cysteine oxidation. The BODIPY/Sypro ruby ratio reflects the oxidation status of cysteine relative to the total protein. The example shows only the partial oxidation of the protein; complete cysteinyl oxidation would result in a very low BODIPY/Sypro ratio, indicating extreme oxidation. BD, BODIPY; Syp, Sypro ruby. (B) A representative image of a 2-D gel of proteins labeled with BODIPY. Nuclear extracts from A549 cells transfected with Prdx-1 siRNA and infected with RSV were labeled with BODIPY and separated by 2-DE. The numbers on the gel indicate individual protein spots whose abundance has changed >1.2-fold. These were identified by electrospray tandem MS on the OrbiTrap Velo. Their identities are listed in Table 2. (C) Expanded view of a few exemplary nuclear protein spots with notable differences in abundances from cells transfected with control, Prdx-1, or Prdx-1 plus Prdx-4 siRNAs.