Figure 4.

Prenylomic methodology and proteomic identification of prenylated proteins labeled with C10AlkOPP or C15AlkOPP. (A) Schematic of prenylomic workflow. Lovastatin pretreated COS7 cells were treated with one of the alkyne probes and the corresponding control, either FPP or farnesol (FOH) for 24 h. Labeled proteins were enriched using a CuAAC reaction with azide-biotin followed by a neutravidin pulldown. After an on-bead digestion three replicates of the control labeled peptides and three alkyne analogue controls were labeled with TMT6Plex for quantification. (B-E) Volcano plots of prenylomic data for C15AlkOPP, C15AlkOH, C10AlkOPP, and C10AlkOH. Volcano plots were generated from a t-test analyzing the normalized intensity for reporter ions for each of the three replicates for each treatment, with an FDR =1% and s⁰=0.5. (B) Plot of C15AlkOPP compared to FPP. (C) Plot of C15AlkOH compared to FOH. (D) Plot of C10AlkOPP compared to FPP. (E) Plot of C10AlkOH compared to FPP. For all plots; red points are FTase substrates, blue points are GGTase I substrates, green points are GGTase II substrates, yellow points are the self-prenylating protein ALDH9A1, and gray points are proteins that are not known to be prenylated. Square points are proteins found with prenylomics for the first time. (F-G) Venn diagrams of comparisons of ungrouped enriched proteins of (F) C15AlkOPP and C15AlkOH or (G) C10AlkOPP and C10AlkOH. H) Correlation Log₂ fold change values for of proteins found both with C15AlkOPP and C10AlkOPP.