INRA, UMR 1331, Toxalim, Research Center in Food Toxicology, FR 31027 Toulouse, France.
Anal Chem. 2013 Sep 3;85(17):8412-20. doi: 10.1021/ac401872f. Epub 2013 Aug 20.
Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins.
由于许多这些分子具有工业应用价值，并且真菌对公共健康和自然生态系统的威胁也日益显现，因此真菌次生代谢产物的特征描述已成为一项挑战。鉴于此，本研究旨在开发一种非靶向方法，通过结合高精度质谱和真菌代谢物的双同位素标记来分析真菌次生代谢产物。烟曲霉 NRRL 35693 是一种重要的真菌病原体，在三种小麦谷物基质上生长：（1）天然富集谷物（99%（12）C），（2）（13）C 富集 96.8%的谷物，（3）（13）C 富集 53.4%和（15）N 富集 96.8%的谷物。通过高效液相色谱-高分辨率质谱（HPLC-HRMS）分析， unambiguously 鉴定了 21 种次生代谢产物。AntiBase 2012 用于确认这些代谢物的身份。此外，基于串联质谱（MS(n)) 实验，首次能够确定 fumigaclavine D 的分子式和结构，fumigaclavines 家族的一个新成员。在 HPLC-HRMS 分析中，通过检测非真菌来源的一个碳原子，还鉴定了 tryptoquivaline F 被甲醇后生物合成降解的产物。该方法的意义不仅在于明确确定真菌次生代谢产物的确切化学公式，还在于易于区分非真菌产物。本研究成功验证了该方法的有效性，现在可以将其应用于其他真菌代谢产物，为发现新的药物或毒素提供了巨大的可能性。
