The purpose of this research is to compare levels of unprenylated Rab proteins in CHM-/- iPSC-RPE cells with and without compactin. Compactin is a statin that inhibits prenyl synthesis and thereby reduces prenylation overall and has an unbiased inhibitory effect on all protein prenylation. So we expect that for Rabs that are already poorly prenylated at baseline in choroideremia RPE cells, compactin will have minimal effect. However, for Rabs that are efficiently prenylated at baseline, compactin should have a much greater effect. And then we used tandem mass tag spectrometry to compare the ratio of each unprenylated Rab in compactin-treated choroideremia cells vs untreated choroideremia cells. In the spreadsheet, "F8" refers to the CHM-/- iPSC-RPE cells and "WT" refers to the isogenic control iPSC-RPE cells. In the "Proteins only" tab, column M shows the ratio of each protein in "DMSO" (untreated) choroideremia cells compared to Compactin-treated choroideremia cells. Compactin-treated control cells are also included in other columns. Untreated control cells could not be used because prenylation is so efficient in these cells, there is almost no material available after doing the in vitro prenylation assay (i.e. almost no unprenylated proteins to biotinylate).
The column descriptions can be found in the sheet titled "Explanations." In addition, AAs= number of amino acids in the protein, MW= molecular weight of the protein, and pI= isoelectric point.
The software is set to report abundance values only when certain criteria are met (S/N of 6, unique peptide etc). A value is NOT reported when the data for a protein fall below these criteria and the cell is instead left blank.
Raeker, M.O., Perera, N.D., Karoukis, A.J., Chen, L., Feathers, K.L., Ali, R.R., Thompson, D.A., Fahim, A.T. Reduced retinal pigment epithelial autophagy due to loss of Rab12 prenylation in a human iPSC-RPE model of choroideremia. Cells, manuscript accepted, in press.
