The isoprenoids farnesyl-(FPP) and geranylgeranylpyrophosphate (FPP and GGPP) are two major lipid intermediates in the mevalonate pathway. They participate in post-translational modification of members of the superfamily of small guanosine triphosphatases (GTPases; Ras, Rab, Rac, etc.) via prenylation reactions. Due to the important role of these proteins in a number of cell processes, in particular cell growth, division, and differentiation, investigation of the involvement of isoprenoids in these processes is of great interest. In a previously published report, we described a fully validated assay for the quantitation of the two isoprenoids using a high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) method. The current work expands on the previous method and enhances it greatly by using a much faster state-of-the-art ultrahigh-performance liquid chromatography (UHPLC) technique coupled to tandem mass spectrometry (MS/MS). The method exhibited a linear concentration range of 5-250 ng/mL for FPP and GGPP in human brain tissue; it was shown to be unaffected by ion suppression and provided results almost six times faster than the HPLC-FLD assay. Comparison of UHPLC-MS/MS and HPLC-FLD yielded excellent comparability of the two assays for both isoprenoids. Based on the UHPLC-MS/MS assay, a novel in vitro test system was implemented to study enzyme specificity for distinct amino acid CAAX motifs, which is potentially useful for investigating target interactions of new therapeutics for diseases involving pathological regulation of isoprenoids and/or small GTPases.
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Acknowledgements The authors would like to thank the Hanna Bragard-Apfel Foundation, the LA Brain Bank, and the Medical Research Council for support. WGW acknowledges the provision of AFI grant #08823 and NIH grants AG23524 and AG18357 as well as the support of the Department of Veterans Affairs. DAV acknowledges research support from the Alfried Krupp von Bohlen und Halbach-Stiftung.
- Brain tissue
- Farnesyl pyrophosphate (FPP)
- Geranylgeranyl pyrophosphate (GGPP)
- Post-translational modification (PTM)
- Prenylation kinetics
- Tandem mass spectrometry
- Ultrahigh-performance liquid chromatography (UHPLC)