Miranda-Santos Ines, Gramacho Silvia, Pineiro Marta, Martinez-Gomez Karla, Fritz Michel, Hollemeyer Klaus, Salvador Armindo, Heinzle Elmar
Department of Life Sciences, Faculty of Science and Technology, University of Coimbra , Coimbra 3000-456, Portugal.
Anal Chem. 2015 Jan 6;87(1):617-23. doi: 10.1021/ac503305w. Epub 2014 Dec 18.
Nucleosides are biosynthesized from metabolites that are at key nodes of intermediary metabolism. Therefore, (13)C 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 (13)C metabolic flux analysis as indicated by a labeling experiment applying [1-(13)C]glucose in a yeast culture.
核苷由处于中间代谢关键节点的代谢物生物合成。因此，在经过适当设计的同位素示踪研究中，核糖核酸（RNA）和脱氧核糖核酸（DNA）中核苷的（13）C标记模式可提供有关增殖细胞代谢通量分布的信息。在此，我们提出一种基于气相色谱（GC）-质谱（MS）的方法，该方法能够挖掘这种潜力。为了阐明RNA和DNA中核苷的位置同位素异构体，我们筛选了其三甲基硅烷基衍生物的碎片谱。我们确定了保留在各个碎片离子中的分子离子部分，特别关注碳骨架。在N-糖苷键处断裂的核苷会产生核苷酸碱基和/或核糖或2'-脱氧核糖碎片离子及其片段。还观察到由核苷酸碱基加上核糖环的一些碳组成的核苷片段。我们总共明确地确定了31个碎片。如在酵母培养物中应用[1-（13）C]葡萄糖的标记实验所示，所确定碎片的质量同位素分布为后续的（13）C代谢通量分析提供了有价值的信息。
