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Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics


Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia.

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Fig. 1: Global labelling of prenylated proteins using novel alkyne-tagged isoprenoid analogues.
Fig. 2: Prenylated protein discovery in EA.hy926 cells using prenyl probes YnF and YnGG.
Fig. 3: Direct detection of post-translational processing of prenylated peptides by LC–MS/MS.
Fig. 4: Global in-cell characterization of prenyl transferase inhibitors by quantitative chemical proteomics in EA.hy926 cells.
Fig. 5: Interrogation of alternative prenylation in response to PTI treatment in EA.hy926 cells.
Fig. 6: Rep-1 knockout reduces geranylgeranylation of a subset of Rab proteins in mouse embryonic fibroblasts.

Data availability

All relevant data are available from the authors. The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium ( via the PRIDE partner repository49, with data set identifier PXD009155.


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The authors thank L. Haigh (Department of Chemistry Mass Spectrometry Facility, Imperial College London) for assistance in acquiring nanoLC–MS/MS and high-resolution mass spectrometry (HRMS) data, S. Sheppard and B. Chappell for their contributions to prenyl probe synthesis, N. O’Reilly (Francis Crick Institute) for peptide substrate synthesis and A.I. Magee (Imperial College London) for insightful comments on the manuscript. This study was supported by Cancer Research UK (Programme Foundation Award C29637/A20183 to E.W.T.), the British Heart Foundation (PhD studentship to E.M.S. and Project Grant PG/12/67/29773 to B.W.S. and E.W.T.), the European Union Framework Programme 7 (Marie Curie Intra European Fellowship to J.Mo.-S. and E.W.T.), Wellcome Trust (Programme Grant 093445/Z/10/Z to M.C.S. and WT102871MA to J.Ma.-S.) and a Royal Thai Government scholarship (PhD studentship to N.P.).

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Authors and Affiliations



E.M.S., J.Mo.-S., R.A.S., N.P., B.W.-S. and E.W.T designed the experiments. E.M.S. performed chemical syntheses of reagents, initial method validation and optimization, experiments related to isoprenoid competition and inhibitor treatment, and analysed data. J.Mo.-S. performed experiments related to isoprenoid competition, inhibitor treatment, prenylation dynamics and Rep-1 knockout, and analysed data. R.A.S. performed chemical synthesis of reagents, conducted preliminary experiments and performed proteomic data analysis. N.P. performed chemical synthesis of pyrophosphate derivatives, biochemical enzyme assays, experiments related to concentration-dependent probe incorporation and analysed data. T.L.-H. designed the biochemical enzyme assays and analysed data. T.T. and M.C.S. provided mouse embryonic fibroblasts. L.N.V. and J.Ma.-S. provided ULK3 and CEP85 protein constructs. E.W.T. conceived and directed the study. E.M.S., J.Mo.-S., R.A.S. and E.W.T. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Edward W. Tate.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–29, Supplementary Table 1, and Supplementary Methods.

Supplementary Data 1

Summary of data on 96 prenylated proteins identified in this study, including literature analysis of prior evidence for prenylation.

Supplementary Data 2

Whole proteome analysis of isoprenoid competition versus YnF and YnGG.

Supplementary Data 3

Whole proteome analysis of YnF and YnGG probe concentration gradient

Supplementary Data 4

Whole proteome analysis of prenylated, probe-modified peptides

Supplementary Data 5

Whole proteome analysis of YnF labelling in response to FTI-277, Tipifarnib and Manumycin A

Supplementary Data 6

Whole proteome analysis of YnGG labelling in response to GGTI-2133

Supplementary Data 7

Whole proteome analysis of prenyl probe preference and prenylation switch in response to Tipifarnib

Supplementary Data 8

Whole proteome prenylation analysis in Rep-1 knock-out fibroblasts vs wild type fibroblasts

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Storck, E.M., Morales-Sanfrutos, J., Serwa, R.A. et al. Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics. Nat. Chem. 11, 552–561 (2019).

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