Volume Determination with Two Standards Allows Absolute Quantification and Improved Chemometric Analysis of Metabolites by NMR from Submicroliter Samples
Volume Determination with Two Standards Allows Absolute Quantification and Improved Chemometric Analysis of Metabolites by NMR from Submicroliter Samples
复制标题
使用两种标准品测定体积可以通过 NMR 对亚微升样品中的代谢物进行绝对定量和改进的化学计量分析
作者：
T. J. Ragan;A. Bailey;A. Gould;P. Driscoll
精确测量微小生物样本中的代谢物浓度通常是可取的，但它仍然具有挑战性。在许多情况下，起始分析物体积不是精确已知的，或者不能直接测量，因此很难计算绝对代谢物浓度。在这里，我们介绍了使用两个标准(VDTS)的体积测定作为一种通用的定量方法，使用1H核磁共振光谱分析亚微升样品中的极性代谢物。这种方法允许从未知体积的小生物样品中反向计算绝对代谢物浓度。在小样本量也是可变的情况下，VDTS可以改进多变量化学计量学分析。在这种情况下，当我们使用VDTS计算的体积校正光谱而不是原始光谱的概率商归一化(PQN)时，主成分分析(PCA)产生了更多逻辑上一致和生物上有洞察力的输出。作为原理的证明，基于Vdts的方法和主成分分析方法被用于分析从非常小但广泛使用的遗传模式生物果蝇的幼虫血淋巴(血)中提取的极性代谢物。这一分析表明，当幼虫摄食充足时，雄虫和雌虫的血淋巴代谢物明显不同。然而，当幼虫的食物营养受到限制时，特定性别的代谢物往往会收敛。我们讨论了这些令人惊讶的结果的生物学意义，并将它们与之前对果蝇血淋巴和哺乳动物血浆的分析进行了比较和对比。综上所述，这些发现揭示了果蝇系统代谢物中一种有趣的、迄今未知的性别二型性，显然值得进一步的生物学研究。重要的是，VDTS方法应该适用于许多不同的分析平台，包括质谱学。
The accurate measurement of metabolite concentrations in miniscule biological sample volumes is often desirable, yet it remains challenging. In many cases, the starting analyte volumes are imprecisely known, or not directly measurable, and hence absolute metabolite concentrations are difficult to calculate. Here, we introduce volume determination using two standards (VDTS) as a general quantitative method for the analysis of polar metabolites in submicrolitre samples using 1H NMR spectroscopy. This approach permits the back calculation of absolute metabolite concentrations from small biological samples of unknown volume. Where small sample volumes are also variable, VDTS can improve multivariate chemometric analysis. In this context, principal component analysis (PCA) yielded more logically consistent and biologically insightful outputs when we used volume-corrected spectra, calculated using VDTS, rather than probabilistic quotient normalization (PQN) of raw spectra. As proof-of-principle, the VDTS-based method and PCA were used to analyze polar metabolites in the hemolymph (blood) extracted from larvae of the very small but widely used genetic model organism Drosophila. This analysis showed that the hemolymph metabolomes of males and females are markedly different when larvae are well fed. However, gender-specific metabolomes tend to converge when larval dietary nutrients are restricted. We discuss the biological implications of these surprising results and compare and contrast them to previous analyses of Drosophila hemolymph and mammalian blood plasma. Together, these findings reveal an interesting and hitherto unknown sexual dimorphism in systemic Drosophila metabolites, clearly warranting further biological investigation. Importantly, the VDTS approach should be adaptable to many different analytical platforms, including mass spectrometry.
影响因子：
29
作者：
Tennessen JM;Baker KD;Lam G;Evans J;Thummel CS
通讯作者：
Thummel CS
通过 31P-NMR 光谱观察果蝇幼虫中的酪氨酸-O-磷酸。
DOI：
10.1016/0167-4889(85)90011-4
发表时间：
1985
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Moore,RR;Burt,CT;Roberts,MF
通讯作者：
Roberts,MF