Work Description

Date: 3 July, 2024

Dataset Title: Protein prenylation mass spectrometry data for choroideremia versus control iPSC-RPE cells

Dataset Creators: Abigail T. Fahim

Dataset Contact: Abigail T. Fahim, ahteich@umich.edu

Funding: National Institutes of Health (NIH)

Description:
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.​

Methodology:
Cell lysates from CHM-/- iPSC-RPE cells with and without compactin treatment, and control iPSC-RPE cells with compactin treatment, were used for an in vitro prenylation assay in which biotin groups were added to all proteins undergoing prenylation. Magnetic streptavidin beads were then used to pull down biotin-tagged proteins. Recoverin was added at equal concentration for normalization. Samples underwent Tandem mass tag (TMT) labeling and LC–MS/MS analysis using an Orbitrap Tribid Fusion mass spectrometer. Proteome Discoverer Version 2.1 was used to analyze data.

Date Coverage: 2023-02-01

Instrument and/or Software specifications: NA

Files contained here:
-TMT_Prenylation_Data_for_Cells_Manuscript.xlsx: Spreadsheet with .xslx version of all the sheets below.

-TMT_Prenylation_Data_for_Cells_Manuscript_Explanations.csv: Notes about how to interpret each of the data files.

-TMT_Prenylation_Data_for_Cells_Manuscript_Proteins_Peptides.csv: Rows for each protein with peptide sequences listed below.

-TMT_Prenylation_Data_for_Cells_Manuscript_Proteins_Only.csv: Same info, but without the peptide sequences.

-TMT_Prenylation_Data_for_Cells_Manuscript_TMT_Info.csv: Sample-to-TMT channel info. A description of the 4-day protocol used by the proteomics core to run tandem mass tag mass spectrometry, and which steps were performed on which day. WT#1_COMP1, WT+2_COMP2, and WT#3_COMP3 were 3 samples from control wild-type cells treated with compactin. F8#1_DMSO4, F8#2_DMSO5, F8#3_DMSO6 were 3 samples from CHM-/- cells treated with DMSO. F8#1_COMP7, F8#2_COMP8, and F8#3_COMP9 were 3 samples from CHM-/- cells treated with compactin.

-TMT_Prenylation_Data_for_Cells_Manuscript_PD_2.4_Processing_Info.csv: Information from Proteome Discoverer regarding the processing of the data from this project. This is information generated by the Proteome Discoverer software. Information about these parameters can be found in the Proteome Discoverer User Guide (ThermoFisher).

Related publication(s):
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.

Use and Access:
This data set is made available under a Creative Commons Attribution-Noncommercial license (CC BY-NC 4.0).

To Cite Data:
Fahim, A. T. Protein prenylation mass spectrometry data for choroideremia versus control iPSC-RPE cells [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/hyxk-9388