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.

Figure 1

Metabolic labeling and “click” tagging of geranylgeranylated proteins using the Cu(I)-catalyzed azide–alkyne cycloaddition reaction. (A) Overview of the “GG-azide”-labeling approach for the labeling and detection of geranylgeranylated proteins. (B) Schematic representation of the “click” reaction.

Figure 2

Azide-GG-OH is a substrate for protein geranylgeranylation in vivo. (A) Labeling and detection of geranylgeranylated proteins in Jurkat cells. Jurkat cells were metabolically labeled with DMSO, N₃-GG-OH (G) or N₃-F-OH in the presence of lovastatin (FL, where F stands for N₃-F-OH) for 48 h. (B) and (C) Labeling and detection of geranylgeranylated proteins in other cell lines. Metabolic labeling experiments of COS-7 (B) and MCF-7 (C) were performed using N₃-GG-OH (G), N₃-F-OH (F) in the presence or absence of lovastatin (L) as indicated.

Figure 3

pH Fractionation of TAMRA-labeled, geranylgeranylated proteins in MCF-7 cells followed by 1-D SDS-PAGE. TAMRA-labeled samples were subjected to pH fractionation on the Zoom^® IEF fractionator into five different pH fractions and then resolved by 1-D SDS-PAGE. (lane 1: DMSO, pH 3–4.6; lane 2: N₃-GG-OH, pH 4.6–5.4; lane 3: N₃-GG-OH, pH 5.4–6.2; lane 4: N₃-GG-OH, pH 6.2–7; lane 5: N₃-GG-OH, pH 7–10; lane 6: N₃-GG-, pH 3–4.6; lane 7: DMSO, pH 4.6–5.4; lane 8: DMSO, pH 5.4–6.2; lane 9: DMSO, pH 6.2–7; lane 10: DMSO, pH 7–10.

Figure 4

pH Fractionation of TAMRA-labeled, geranylgeranylated proteins in MCF-7 cells followed by 2-D SDS-PAGE. TAMRA-labeled samples were pH fractionated by the Zoom^® IEF fractionator into different pH fractions, followed by narrow pH range 2-D SDS-PAGE. TAMRA-labeled, geranylgeranylated proteins were visualized by TAMRA-labeled protein detection.

Figure 5

Examination of prenylation of progerin, a mutant form of prelamin A. Fibroblasts from mice expressing geranylgeranylated progerin (LmnaggHG/+), farnesylated progerin (LmnaHG/+), or mature lamin A (Lmna+/+) were metabolically labeled with N₃-GG-OH for 48h. Geranylgeranylated, TAMRA-labeled proteins were visualized with a fluorescence imager. Separated proteins were also detected by Western blotting using an antibody against lamin A/C.