Abstract
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.
Abbreviated mevalonate/isoprenoid/cholesterol pathway. 3-HMG-CoA reductase activity leads to mevalonate (MVA), which is a precursor of farnesyl pyrophosphate (FPP) formed by FPP synthase. FPP is a branching point and serves as a precursor of geranylgeranyl pyrophosphate (GGPP) by activation of GGPP synthase. Both FPP and GGPP are involved in post-translational modification of proteins (protein prenylation). FPP is also the precursor of cholesterol
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cole SL, Vassar R (2006) Isoprenoids and Alzheimer’s disease: a complex relationship. Neurobiol Dis 22:209–222
Hooff GP, Volmer DA, Wood WG, Muller WE, Eckert GP (2008) Isoprenoid quantitation in human brain tissue: a validated HPLC–fluorescence detection method for endogenous farnesyl-(FPP) and geranylgeranylpyrophosphate (GGPP). Anal Bioanal Chem 392:673–680
Hooff GP, Wood WG, Muller WE, Eckert GP (2010) Isoprenoids, small GTPases and Alzheimer’s disease. Biochim Biophys Acta 1801:896–905
McTaggart SJ (2006) Isoprenylated proteins. Cell Mol Life Sci 63:255–267
Winter-Vann AM, Casey PJ (2005) Post-prenylation—processing enzymes as new targets in oncogenesis. Nat Rev Cancer 5:405–412
Sousa SF, Fernandes PA, Ramos MJ (2008) Farnesyl transferase inhibitors: a detailed chemical view on an elusive biological problem. Curr Med Chem 15:1478–1492
Yokoyama K, Zimmerman K, Scholten J, Gelb MH (1997) Differential prenyl pyrophosphate binding to mammalian protein geranylgeranyl transferase-I and protein farnesyl transferase and its consequence on the specificity of protein prenylation. J Biol Chem 272:3944–3952
Eckert GP, Hooff GP, Strandjord DM, Igbavboa U, Volmer DA, Muller WE, Wood WG (2009) Regulation of the brain isoprenoids farnesyl- and geranylgeranyl pyrophosphate is altered in male Alzheimer patients. Neurobiol Dis 35:251–257
Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701
Karnoub AE, Weinberg RA (2008) Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 9:517–531
Alegret M, Silvestre JS (2006) Pleiotropic effects of statins and related pharmacological experimental approaches. Methods Find Exp Clin Pharmacol 28:627–656
Capell BC, Erdos MR, Madigan JP, Fiordalisi JJ, Varga R, Conneely KN, Gordon LB, Der CJ, Cox AD, Collins FS (2005) Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson–Gilford progeria syndrome. Proc Natl Acad Sci U S A 102:12879–12884
Tong H, Wiemer AJ, Neighbors JD, Hohl RJ (2008) Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue. Anal Biochem 378:138–143
US Department of Health and Human Services, Food and Drug Administration (2001) Guidance for industry—bioanalytical method validation. Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), pp 1–22
Lebowitz PF, Casey PJ, Prendergast GC, Thissen JA (1997) Farnesyl transferase inhibitors alter the prenylation and growth-stimulating function of RhoB. J Biol Chem 272:15591–15594
Carboni JM, Yan N, Cox AD, Bustelo X, Graham SM, Lynch MJ, Weinmann R, Seizinger BR, Der CJ, Barbacid M (1995) Farnesyl transferase inhibitors are inhibitors of Ras but Not R-Ras2/TC21, transformation. Oncogene 10:1905–1913
Armstrong SA, Hannah VC, Goldstein JL, Brown MS (1995) CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem 270:7864–7868
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hooff, G.P., Patel, N., Wood, W.G. et al. A rapid and sensitive assay for determining human brain levels of farnesyl-(FPP) and geranylgeranylpyrophosphate (GGPP) and transferase activities using UHPLC–MS/MS. Anal Bioanal Chem 398, 1801–1808 (2010). https://doi.org/10.1007/s00216-010-4088-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-010-4088-7