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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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.).
The authors declare no competing interests.
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Supplementary Figures 1–29, Supplementary Table 1, and Supplementary Methods.
Summary of data on 96 prenylated proteins identified in this study, including literature analysis of prior evidence for prenylation.
Whole proteome analysis of isoprenoid competition versus YnF and YnGG.
Whole proteome analysis of YnF and YnGG probe concentration gradient
Whole proteome analysis of prenylated, probe-modified peptides
Whole proteome analysis of YnF labelling in response to FTI-277, Tipifarnib and Manumycin A
Whole proteome analysis of YnGG labelling in response to GGTI-2133
Whole proteome analysis of prenyl probe preference and prenylation switch in response to Tipifarnib
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). https://doi.org/10.1038/s41557-019-0237-6
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