Abstract
Certain mammalian proteins are modified at a carboxyl-terminal cysteine by a thioether-linked prenyl group, the 15-carbon farnesyl or the 20-carbon geranylgeranyl moiety. Here, we describe analytical methods to determine the presence of a prenyl modification, the structure of the prenyl group, and the lipid: protein molar ratio for candidate proteins or their proteolytic fragments. Methods for the synthesis of prenyl standards are also presented. Methyl iodide or Raney nickel treatment is used to release the prenyl group from the protein for further analysis. When the prenyl group has been radiolabeled biosynthetically, two analytical techniques are available. Methyl iodide-released material, primarily the prenyl alcohol, can be cochromatographed with known standards using reverse-phase high-performance liquid chromatography to provide indirect structural data. Raney nickel-released material, primarily the unsubstituted hydrocarbon, can be analyzed by radiometric gas chromatography, which offers more precise structural information. However, unequivocal determination of the structure and quantitation of the mass of the released prenyl compound requires the use of gas chromatography-coupled mass spectrometry.
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