Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.
Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories.
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DOI：
10.1111/gbi.12132
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发表时间：
2015-07
期刊：
影响因子：
3.7
通讯作者：
Newman DK
中科院分区：
文献类型：
--
作者：
Wu CH;Kong L;Bialecka-Fornal M;Park S;Thompson AL;Kulkarni G;Conway SJ;Newman DK
类霍帕酸是来自类异戊二烯家族的类固醇样脂质，主要由细菌产生。Hopanes是Hopanoids的分子化石，如果它们的来源和生物学功能可以被限制，那么它们提供了洞察早期地球环境转变的潜力。在过去的二十年中，已经开发了用于从培养物和环境样品中质谱分析类Hopanoids的半定量方法。然而，hopanoids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的差异，迄今为止已经排除了可靠的定量，并阻碍了实验室之间结果的比较。这些电离不一致性导致需要用纯化的类霍帕酸校准单个仪器以可靠地定量类霍帕酸。在这里，我们提出了新的方法来获得纯化和合成的定量标准。我们优化了Rhodopterium palustris TIE-1中2-甲基藿烷类化合物的生产，并纯化了2 Me-diplopterol、2 Me-bacteriophanetetrol（2 Me-BHT）及其未甲基化的物质（diplopterol和BHT）。我们发现，2-甲基化使diplopterol的信号强度降低2 - 34%，这取决于用于检测它的仪器，但使BHT信号降低不到5%。此外，2 Me-diplopterol产生的离子计数比等量的2 Me-BHT高10倍。在GC中使用火焰离子化检测器进行信号定量也观察到类似的偏差。然而，在LC-MS中，2 Me-BHT产生的离子计数比2 Me-diplopterol高11倍，但离子计数仅比甾醇标准品乙酸异丙酯高1.2倍。为了进一步提高定量，我们合成了四氘代（D4）diplopterol，一种用于各种hopanoids的前体。对（D4）-双普斯特罗和磷脂混合物的LC-MS分析表明，在共洗脱磷脂的影响下，D4-双普斯特罗内标物比外标物更准确地定量双普斯特罗。这些新的定量方法允许研究之间进行有意义的比较，从而在实验室和环境样品中进行更准确的hopanoid模式检测。
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.