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Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.
Geobiology ( IF 2.7 ) Pub Date : 2015-04-11 , DOI: 10.1111/gbi.12132
C-H Wu 1, 2 , L Kong 3 , M Bialecka-Fornal 1 , S Park 2 , A L Thompson 3 , G Kulkarni 1 , S J Conway 3 , D K Newman 1, 2, 4
Affiliation  

Hopanoids are steroid‐like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2‐methylhopanoid production in Rhodopseudomonas palustris TIE‐1 and purified 2Me‐diplopterol, 2Me‐bacteriohopanetetrol (2Me‐BHT), and their unmethylated species (diplopterol and BHT). We found that 2‐methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me‐diplopterol produces 10× higher ion counts than equivalent quantities of 2Me‐BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC‐MS, however, 2Me‐BHT produces 11× higher ion counts than 2Me‐diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D4) diplopterol, a precursor for a variety of hopanoids. LC‐MS analysis on a mixture of (D4)‐diplopterol and phospholipids showed that under the influence of co‐eluted phospholipids, the D4‐diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.

中文翻译:

定量类胡hop分析可实现强大的模式检测和实验室之间的比较。

类胡萝卜素是类异戊二烯家族的类固醇类脂质,主要由细菌产生。ane烷是of烷的分子化石,如果它们的来源和生物学功能受到限制,则可能提供洞悉早期地球环境转变的潜力。在过去的二十年中,已经开发出了半定量方法,用于对来自文化和环境样品的类hop蛇类化合物进行质谱分析。然而,迄今为止,类胡萝卜素的结构多样性以及它们在不同仪器上的电离效率可能存在差异,因此至今仍无法进行可靠的定量分析,并妨碍了实验室之间结果的比较。这些电离不一致导致需要使用纯化的类胡萝卜素校准各个仪器以可靠地定量类胡萝卜素。这里,我们提出了获得纯化和合成定量标准品的新方法。我们优化了2-甲基类黄酮的生产拟南芥(Rhodopseudomonas palustris) TIE-1和纯化的2Me-diplopterol,2Me-bacteriohopanetetrol(2Me-BHT)及其未甲基化物种(diplopterol和BHT)。我们发现2-甲基化可将二蝶呤醇的信号强度降低2%至34%,具体取决于用于检测它的仪器,但将BHT信号的强度降低到5%以下。此外,2Me-双蝶呤醇产生的离子数比2Me-BHT的当量高10倍。使用火焰离子化检测器在GC中进行信号定量也观察到了类似的偏差。然而,在LC-MS中,2Me-BHT产生的离子数比2Me-双蝶呤醇高11倍,但比固醇标准醋酸戊酯高出1.2倍。为了进一步改善定量分析,我们合成了四氘代(D 4)二蝶呤醇,一种多种类蛇药的前体。对(D 4)-双蝶呤和磷脂的混合物进行LC-MS分析表明,在共洗脱磷脂的影响下,D4-双蝶呤的内标比对双蝶呤的内标更准确地定量。这些新的定量方法可以在研究之间进行有意义的比较,从而可以在实验室和环境样品中更准确地检测出类hop蛇图案。
更新日期:2015-04-11
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