Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry

作者：Jauhiainen Marjo^*; Monkkonen Hannu; Raikkonen Johanna; Monkkonen Jukka; Auriola Seppo

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2009, 877(27): 2967-2975.

DOI：10.1016/j.jchromb.2009.07.010

• ATP analogs
• Bisphosphonates
• Mevalonate pathway
• Zoledronic acid

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, Apppl (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. Apppl has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC-MS(2) for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030 mu M (1.35 fmol on-column) and for Apppl and ApppD 0.020 mu M (0.9 fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS(2) were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS(3) spectra were followed. IPP and DMAPP accumulation as well as Apppl and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25 mu M zoledronic acid (an N-BP) for 24 h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4 nmol/mg of protein and amount of Apppl and ApppD was 1.1 nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain I PP, DMAPP, Apppl or ApppD.

• 出版日期2009-10-1