Important Requirements for Desorption/Ionization Mass Spectrometric Measurements of Temozolomide-Induced 2′-Deoxyguanosine Methylations in DNA

Abstract

In clinical pharmacology, drug quantification is mainly performed from the circulation for pharmacokinetic purposes. Finely monitoring the chemical effect of drugs at their chemical sites of action for pharmacodynamics would have a major impact in several contexts of personalized medicine. Monitoring appropriate drug exposure is particularly challenging for alkylating drugs such as temozolomide (TMZ) because there is no flow equilibrium that would allow reliable conclusions to be drawn about the alkylation of the target site from plasma concentrations. During the treatment of glioblastoma, it appears, therefore, promising to directly monitor the alkylating effect of TMZ rather than plasma exposure, ideally at the site of action. Mass spectrometry (MS) is a method of choice for the quantification of methylated guanines and, more specifically, of O6-methylguanines as a marker of TMZ exposure at the site of action. Depending on the chosen strategy to analyze modified purines and 2′-deoxynucleosides, the analysis of methylated guanines and 2′-deoxyguanosines is prone to important artefacts due to the overlap between masses of i) guanines from DNA and RNA, and ii) different methylated species of guanines. Therefore, the specific analysis of O6-methyl-2′deoxyguanosine, which is the product of the TMZ effect, is highly challenging. In this work, we report observations from matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray ionization (DESI) MS analyses. These allow for the construction of a decision tree to initiate studies using desorption/ionization MS for the analysis of 2′-deoxyguanosine methylations induced by TMZ.

Quantification of Biologically Active DNA Alkylation in Temozolomide-Exposed Glioblastoma Cell Lines by Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry: Method Development and Recommendations for Validation

“…When analyzing methylated guanines, the presence of alkylated RNA prevents the specific quantification of methylated guanines from DNA without a sample preparation including RNA removal. , Since reducing sample preparation steps would be critical for future sensitive analysis of low-volume samples (e.g., low cell counts of limited tissue pieces), analysis of O6-m2dGO would thus be the most suited strategy to potentially skip the RNA removal step. We have recently demonstrated that using desorption/ionization MS methods would only be possible with instruments equipped with ion mobility and specific high-resolution (HR) settings that would efficiently separate O6 and N7 species of methylated guanines and 2dGO . In this context, the analysis of nucleosides by ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) seems to be the most appropriate method to separate O6- and N7-methylated species based on their difference of hydrophobicity.…”

“…We have recently demonstrated that using desorption/ionization MS methods would only be possible with instruments equipped with ion mobility and specific high-resolution (HR) settings that would efficiently separate O6 and N7 species of methylated guanines and 2dGO. 3 In this context, the analysis of nucleosides by ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) seems to be the most appropriate method to separate O6- and N7-methylated species based on their difference of hydrophobicity. Analyses in multiple reaction monitoring (MRM) mode allow for the selection and fragmentation of compounds with high specificity.…”