Quantitative analysis of 5'-deoxy-5'-methylthioadenosine in melanoma cells by liquid chromatography-stable isotope ratio tandem mass spectrometry.
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.
Stevens
Axel P
AP
Institute of Functional Genomics, University of Regensburg, Josef-Engert-Strasse 9, Regensburg, Germany.
Dettmer
Katja
K
Wallner
Susanne
S
Bosserhoff
Anja K
AK
Oefner
Peter J
PJ
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D003839:Deoxyadenosines / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000737:chemistry*; D011683:Purine-Nucleoside Phosphorylase / Q000378:metabolism; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013872:Thionucleosides / Q000032:analysis* / Q000378:metabolism
D003839:Deoxyadenosines; D013872:Thionucleosides; C008500:5'-methylthioadenosine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase