[40] Isoprenylation of γ subunits and G-protein effectors

Several approaches have been developed for the identification of isoprenyl groups on modified proteins. The first, and most rigorous, involves chemical cleavage of the modifying isoprenyl group with Raney nickel, followed by gas chromatography-coupled mass spectrometric (GC–MS) analysis of the products. The advantage of GC–MS analysis is that definitive identification of the isoprenyl group, as well as its stereochemical configuration, can be determined. This chapter describes a detailed protocol for the GC–MS analysis of isoprenylated proteins. There are also two variations of this approach. The first involves the release of the modifying isoprenoid with methyl iodide, followed by the separation of the resulting products by high-performance liquid chromatography (HPLC) and structural analysis by GC–MS. This method, however, does not allow the rigorous assignment of the stereochemistry of the modifying isoprenyl group. The second variation employs atom bombardment mass spectrometry to determine the combined mass value of the isoprenylated polypeptide. This information is then used to deduce the identity of the isoprenyl group if the exact amino acid sequence of the polypeptide is known.