A tagging-via-substrate technology for detection and proteomics of farnesylated proteins

Abstract

A recently developed proteomics strategy, designated tagging-via-substrate (TAS) approach, is described for the detection and proteomic analysis of farnesylated proteins. TAS technology involves metabolic incorporation of a synthetic azido-farnesyl analog and chemoselective derivatization of azido-farnesyl-modified proteins by an elegant version of Staudinger reaction, pioneered by the Bertozzi group, using a biotinylated phosphine capture reagent. The resulting protein conjugates can be specifically detected and/or affinity-purified by streptavidin-linked horseradish peroxidase or agarose beads, respectively. Thus, the technology enables global profiling of farnesylated proteins by enriching farnesylated proteins and reducing the complexity of farnesylation subproteome. Azido-farnesylated proteins maintain the properties of protein farnesylation, including promoting membrane association, Ras-dependent mitogen-activated protein kinase kinase activation, and inhibition of lovastatin-induced apoptosis. A proteomic analysis of farnesylated proteins by TAS technology revealed 18 farnesylated proteins, including those with potentially novel farnesylation motifs, suggesting that future use of this method is likely to yield novel insight into protein farnesylation. TAS technology can be extended to other posttranslational modifications, such as geranylgeranylation and myristoylation, thus providing powerful tools for detection, quantification, and proteomic analysis of posttranslationally modified proteins.

Fig. 1.

Schematic representation of the TAS technology for solid-phase affinity isolation of azide-labeled farnesylated proteins. (A) Farnesyltransferase-catalyzed modification of Ras. (B) Chemical structures of F-OH, F-azide-OH, natural FPP, and an FPP-azide. (C) The Staudinger conjugation reaction between bPPCR and an azide-containing molecule. (D) Schematic diagram for the conjugation reaction between a F-azide-modified protein and bPPCR. (E) Experimental procedure for the detection of F-azide modified proteins. Proteins 1 and 2 represent unmodified proteins; protein 3 represents a protein modified by a natural farnesyl group; protein 4 represents an F-azide-modified protein; and bPPCR represents the biotinylated phosphine capture reagent. Only F-azide-modified protein 4 is captured and subsequently detected.

Fig. 2.

Detection and verification of F-azide modification in vivo. F-azide-modified Ras (A) and Hdj-2 (B) were detected by mobility-shift assays. COS-1 cells were labeled with the indicated compounds for 24 h; the cell lysate was resolved by SDS/PAGE and probed by using anti-Ras or anti-Hdj-2 antibodies. Unmodified proteins are indicated by “u,” and farnesylated proteins are indicated by “p.” (C) Confirmation of F-azide modification by reciprocal immunoprecipitation. (D) Global detection of F-azide-modified proteins by Western blotting analysis. The protein lysates from cells with or without metabolic incorporation of FPP-azide were conjugated to bPPCR; the resulting biotinylated proteins were resolved by SDS/PAGE and detected by Western blotting analysis using HRP-conjugated streptavidin. (E) Mobility-shift assay of Rap1. COS-1 cells were labeled with the indicated compounds for 24 h; the cell lysate was resolved by SDS/PAGE and probed by using anti-Rap1 antibody. Unmodified proteins are indicated by “u,” and prenylated proteins are indicated by “p.” The results suggest that FPP azide could not be incorporated into Rap1, a known geranylgeranylated protein.

Fig. 3.

Restoration of membrane association of H-Ras to lovastatin-treated COS-1 cells. COS-1 cells were treated with 20 μM lovastatin alone or together with F-azide-OH (20 μM, lanes 5 and 6) or FPP-azide (20 μM, lanes 7 and 8) for 48 h. Membrane (M) or soluble (S) fractions were separated by ultracentrifugation. Equivalent proportions of membrane and soluble fractions were loaded in each lane. (A) Blotted proteins were probed with H-Ras antibody. (B) Another blot of the same samples was probed with RhoGDI antibody and Na⁺/K⁺ATPase antibody to demonstrate separation of soluble and membrane proteins, respectively.

Fig. 4.

Azido-farnesyl substrates restore the ability of Ras to activate Raf/MAP kinase signaling. (A) Signal transduction cascade of EGF stimulation. (B) Western blotting analysis of Hdj-2, H-Ras, MEK, and phosphorylated MEK using antibodies. COS-1 cells were starved for 48 h in serum-free DMEM. At the same time, the cells were treated with 20 μM lovastatin alone or together with 20 μM azido-farnesyl substrate. After starvation, the COS-1 cells were stimulated with 10 ng/ml EGF, which activates the Raf/MAP kinase cascade through a Ras-dependent pathway. MEK activation was detected by using an antiphospho-specific MEK antibody.

Fig. 5.

An example of nano-HPLC/MS/MS analysis for protein identification. (A) Total ion current chromatogram of a nano-HPLC/MS/MS of the beads digest. (B) MS/MS spectrum of 692.9 m/z at the retention time of 40.67 min, which identified the peptide QGVDDAFYTLVR, unique to K-Ras.