Quantitative analysis of 5'-deoxy-5'-methylthioadenosine in melanoma cells by liquid chromatography-stable isotope ratio tandem mass spectrometry.

Abstract

The frequent deletion of the human chromosomal region 9p21, including the methylthioadenosine phosphorylase (MTAP) gene, is hypothesized to lead to the intra- and/or extracellular accumulation of 5'-deoxy-5'-methylthioadenosine (MTA) in cancer cells and the subsequent promotion of tumor progression. The lack of sensitive methodology for the direct measurement of MTA in tumor cells has hampered the testing of this hypothesis to date. A liquid chromatography electrospray ionization tandem mass spectrometry method (LC-MS/MS) was developed for the absolute quantitative determination of MTA in cell culture media and cell extracts using stable isotope labeled MTA as an internal standard. Limit of detection (LOD) and lower limit of quantification (LLOQ) were 62.5 pM and 2 nM, respectively, and allowed the direct measurement of MTA in biological samples without prior enrichment. Average imprecision of MTA extraction from cells and cell media, as well as LC-MS/MS analysis were 9.7, 3.8 and 1.9%, respectively. The method enabled the demonstration of the accumulation of MTA in melanoma cell culture media reaching a steady-state level within 24h. Only a slight difference in extracellular MTA concentrations was observed between cells with and without MTAP expression. However, there was a fourfold increase in intracellular MTA concentration in melanoma cells lacking MTAP, thus confirming the hypothesized accumulation of MTA in human cancer cells harboring a chromosome 9p21 deletion.

• Authors: Axel P Stevens, Katja Dettmer, Susanne Wallner, Anja K Bosserhoff, Peter J Oefner
• Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 876 Issue 1 Pg. 123-8 (Dec 01 2008) ISSN: 1570-0232 [Print] Netherlands
• PMID: 18996776 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Deoxyadenosines
• Thionucleosides
• 5'-methylthioadenosine
• Purine-Nucleoside Phosphorylase
• 5'-methylthioadenosine phosphorylase

Topics

• Cell Line, Tumor
• Chromatography, Liquid (methods)
• Deoxyadenosines (analysis, metabolism)
• Humans
• Melanoma (chemistry)
• Purine-Nucleoside Phosphorylase (metabolism)
• Sensitivity and Specificity
• Spectrometry, Mass, Electrospray Ionization (methods)
• Tandem Mass Spectrometry (methods)
• Thionucleosides (analysis, metabolism)