A Quantitative Chemical Proteomics Approach Using a Novel Two-Probe System Enables System-Wide Analysis of Protein Prenylation

Proteins prenylation, the post-translational attachment of a farnesyl or geranylgeranyl isoprenoid to one or more C-terminal cysteine residues, is an important modulator of localization and function of proteins such as the Ras isoforms, and a widely studied therapeutic target in number of cancer and other diseases. Despite its clinical importance, tools to interrogate prenylation and to quantify changes in response to treatment or disease on a global scale are lacking. Herein we report the development of two novel isoprenoid analogues, YnF and YnGG, which when used in combination with quantitative proteomics technologies enables the global profiling of prenylated proteins in living cells. The workflow enabled validation of a 51 prenylated CXXX-motif proteins, including seven novel farnesylated substrates, and 29 Rab proteins. Furthermore, we present tools which enable the detection of prenylated peptides at native abundance. We developed a quantitative strategy to decipher changes in prenylation in response to several inhibitors, including the clinically relevant farnesyl transferase inhibitor Tipifarnib, enabling in-cell dose responses of individual inhibitors, as well as shedding light on the alternative prenylation dynamics caused by inhibition of one prenyl transferase. Finally, we show how our methodology can be employed to further our understanding of prenylation in disease models such as Choroideremia by quantifying the effect of prenylation on 30 Rab substrates in response to Rep-1 knock-out.