A novel approach to tag and identify geranylgeranylated proteins

Abstract

A recently developed proteomic strategy, the “GG-azide”-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the “click” chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG-azide”-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications.

Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform

“…1D and Tables S1 and S2). In comparison with previous proteomic studies that targeted subsets of S-prenylated proteins (19)(20)(21)23), our proteomic analysis of alk-FOH-labeled proteins recovered both farnesylated and geranylgeranylated proteins, as well as many other candidate isoprenoid-modified proteins (Tables S1 and S2). To validate our alk-FOH-labeled proteins in our dataset, we biochemically characterized a canonical CaaX-containing farnesylated protein (DnaJA2) and an unpredicted substrate (Pcbp1).…”

“…For example, a biotin geranyl diphosphate analog can be used with engineered prenyltransferases to visualize and profile geranylgeranylated proteins in vitro (19). Alternatively, azide-derivatives of farnesol or geranylgeraniol or their diphosphate analogs can be used to detect prenylated proteins after bioorthogonal labeling with fluorescent or affinity tags (20,21). More recently, alkynyl-isoprenoids have also been developed to enable more efficient labeling and detection of prenylated proteins in mammalian cells compared with their azide-counterparts using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (22,23).…”

“…More recently, alkynyl-isoprenoids have also been developed to enable more efficient labeling and detection of prenylated proteins in mammalian cells compared with their azide-counterparts using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (22,23). The application of these lipid chemical reporters has enabled the enrichment and identification of farnesylated (20) or geranylgeranylated protein subsets (19,21), but the general proteomic coverage of prenylated proteins has been limited. Using an alkyne-farnesol chemical reporter and improved bioorthogonal proteomic methods, we identified over 100 candidate isoprenoidlabeled proteins in macrophages, including small GTPases as well as previously unannotated S-prenylated substrates such as the zinc-finger antiviral protein (ZAP).…”

An Activatable Fluorescent γ-Polyglutamic Acid Complex for Sentinel Lymph Node Imaging

“…28,29) To obtain the effective FRET quenching, the absorption spectrum of the quencher should overlap the emission spectrum of the fluorophore and when the distance between quencher and fluorophore is typically less than 100 . 30) BHQ2 and QSY7 are non-fluorescent dyes, and have absorption peaks from 550 to 650 nm and 500 to 600 nm, respectively.…”

The Impact of Extracellular Low pH on the Anti-Tumor Efficacy Against Mesothelioma

“…It remains unclear yet why statins inhibit cellular proliferation preferentially in tumor growth. Recently, a novel approach to identify geranylgeranylated protein was developed using azide binding geranylgeraniol which converted to geranylgeranyl pyrophosphate in cytosol (Chan et al, 2009). So the proteins, which play a critical role for mesothelioma proliferation at low pH, will be identified soon.…”