Lane Andrew N, Arumugam Sengodagounder, Lorkiewicz Pawel K, Higashi Richard M, Laulhé Sébastien, Nantz Michael H, Moseley Hunter N B, Fan Teresa W-M
Center for Regulatory and Environmental Analytical Metabolomics, University of Louisville, Louisville, KY, USA; J.G. Brown Cancer Center, University of Louisville, Louisville, KY, USA.
Magn Reson Chem. 2015 May;53(5):337-43. doi: 10.1002/mrc.4199. Epub 2015 Jan 23.
NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl-selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating (15)N in the aminooxy functional group, (15)N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here, we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses, for example. Utilizing the 2-bond or 3-bond (15)N-(1)H couplings, the (15)N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl-containing compounds at NMR-detectable levels, six of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts.
从细胞或组织中提取的代谢物混合物的核磁共振波谱极其复杂，这反映了大量化合物以广泛的浓度存在。尽管多维核磁共振可以极大地提高分辨率并提高化合物归属的可靠性，但低丰度代谢物往往仍难以被检测到。我们开发了一种羰基选择性氨氧基探针，它能与游离的酮基和醛基特异性反应，但不与羧酸盐反应。通过在氨氧基官能团中引入（15）N，（15）N编辑的核磁共振被用于专门选择那些含有游离羰基官能团的代谢物，而排除所有其他代谢物。在这里，我们证明了酮和醛的氨氧基加合物的化学位移非常不同，例如可用于区分醛糖和酮糖。利用2键或3键（15）N-（1）H偶合，首先用标准品优化（15）N编辑的核磁共振分析，然后将其应用于肺腺癌细胞系A549的提取物。在核磁共振可检测水平上有30多种含羰基化合物，其中六种我们已通过参考我们的数据库进行了归属。由于氨氧基探针含有一个带永久电荷的季铵基团，加合物也针对质谱检测进行了优化。因此，这种样品制备技术在这两种结构测定工具之间建立了更好的联系，从而为直接在粗生物提取物中更快、更可靠地鉴定已知和未知代谢物铺平了道路。
