Mass Isotopomer Analysis of Nucleosides Isolated from RNA and DNA Using GC/MS
2015
Miranda-Santos, Ines | Gramacho, Silvia | Pineiro, Marta | Martinez-Gomez, Karla | Fritz, Michel | Hollemeyer, Klaus | Salvador, Armindo | Heinzle, Elmar
Nucleosides are biosynthesized from metabolites that are at key nodes of intermediary metabolism. Therefore, ¹³C labeling patterns in nucleosides from ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) in suitably designed isotopic tracer studies provide information on metabolic flux distributions of proliferating cells. Here, we present a gas chromatography (GC)-mass spectrometry (MS)-based approach that permits one to exploit that potential. In order to elucidate positional isotopomers of nucleosides from RNA and DNA, we screened the fragmentation spectra of their trimethylsilyl derivatives. We identified the molecular ion moieties retained in the respective fragment ions, focusing particularly on the carbon backbone. Nucleosides fragmented at the N-glycosidic bond provide nucleobase and/or ribose or 2′-deoxyribose fragment ions and fragments thereof. Nucleoside fragments composed of the nucleobase plus some carbons of the ribose ring were also observed. In total, we unequivocally assigned 31 fragments. The mass-isotopic distribution of the assigned fragments provides valuable information for later ¹³C metabolic flux analysis as indicated by a labeling experiment applying [1-¹³C]glucose in a yeast culture.
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