Figure 6.

In Vivo Characterization of His₆-GFP-BD-CVIL Isoprenylation by MS Analysis of His₆-GFP-BD-CVIL–Derived Peptides.

Solubilized total membrane and supernatant fractions from tobacco BY-2 cells induced to express His₆-GFP-BD-CVIL were resolved by SDS-PAGE, and recombinant His₆-GFP-BD-CVIL was cut from the gel, digested with Asp-N, and extracted and fractionated by solid-phase extraction for MALDI-TOF MS peptide mass fingerprinting and MALDI-TOF MS/MS peptide mass sequencing (detailed in Supplemental Figure 1 online). A significant peak was detected at an m/z of 2852.68, which corresponded to the predicted mass of a geranylgeranylated, methylated C-terminal His₆-GFP-BD-CVIL peptide. Peaks at an m/z of 2756.62 or an m/z of 2784.56, which correspond to unprenylated and farnesylated peptides, respectively, were not detected in the absence of inhibitors (Control). However, a significant peak was detected at an m/z of 2756.62 from tobacco BY-2 cells treated with Fos.