Endoplasmic reticulum resident proteins of normal human dermal fibroblasts are the major targets for oxidative stress induced by hydrogen peroxide

The membrane-permeable fluorescein-labelled tyramine conjugate (acetylTyrFluo) was used to identify the proteins of normal human dermal fibroblasts most susceptible to oxidation by hydrogen peroxide [Van der Vlies, Wirtz and Pap (2001) Biochemistry 40, 7783-7788]. By exposing the cells to H2O2 (0.1 mM for 10 min), TyrFluo was covalently linked to target proteins. TyrFluo-labelled and [S-35]Met-labelled cell lysates were mixed and subjected to two-dimensional PAGE. After Western blotting the S-35-labelled proteins were visualized by autoradiography and the TyrFluo-labelled proteins by using anti-fluorescein antibody. The TyrFluo-labelled proteins were matched with the S-35-labelled proteins and identified by comparison with our mastermap of proteins. Protein disulphide isomerase (PDI), IgG-binding protein (BiP), calnexin, endoplasmin and glucose-regulated protein 58 (endoplasmic reticulum protein 57/GRP58) were identified as targets of oxidation. All these proteins reside in the endoplasmic reticulum and are part of the protein folding machinery. In agreement, confocal laser scanning microscopy showed colocalization of TyrFluo-labelled proteins and the KDEL receptor ERD-2, a marker for the endoplasmic reticulum.