A Azzollini

Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography–time-of-flight mass spectrometry fingerprinting

Access to new biological sources is a key element of natural product research. A particularly large number of biologically active molecules have been found to originate from microorganisms. Very recently, the use of fungal co-culture to activate the silent genes involved in metabolite biosynthesis was found to be a successful method for the induction of new compounds. However, the detection and identification of the induced metabolites in the confrontation zone where fungi interact remain very challenging. To tackle this issue, a high-throughput UHPLC-TOF-MS-based metabolomic approach has been developed for the screening of fungal co-cultures in solid media at the petri dish level. The metabolites that were overexpressed because of fungal interactions were highlighted by comparing the LC-MS data obtained from the co-cultures and their corresponding mono-cultures. This comparison was achieved by subjecting automatically generated peak lists to statistical treatments. This strategy has been applied to more than 600 co-culture experiments that mainly involved fungal strains from the Fusarium genera, although experiments were also completed with a selection of several other filamentous fungi. This strategy was found to provide satisfactory repeatability and was used to detect the biomarkers of fungal induction in a large panel of filamentous fungi. This study demonstrates that co-culture results in consistent induction of potentially new metabolites.

Preparative Scale MS-Guided Isolation of Bioactive Compounds Using High-Resolution Flash Chromatography: Antifungals from Chiloscyphus polyanthos as a Case Study

In natural product research, the efficient purification of molecules from large amounts of complex extracts is a key element. In this regard, an integrative strategy for efficient MS-guided isolation of antifungal compounds has been developed. First, off-line HPLC antifungal activity-based profiling and HPLC-PDA-MS profiling were used to localize the compounds of interest on the analytical scale. Then, the analytical gradient was geometrically transferred to the flash chromatographic level. Finally, an MS-triggered isolation of the localized bioactive molecules was realized using high-resolution flash chromatographic columns (15 µm spherical particles) coupled to a single quadrupole mass spectrometer via a splitter system. This isolation strategy was applied for the MS-targeted purification of antifungal principles from the liverwort Chiloscyphus polyanthos. This rational methodology has high potential for the targeted large-scale purification of bioactive compounds, avoiding the need to repeat a given bioassay at each isolation step. Seven sesquiterpene lactones were isolated, of which five were found to be bioactive and one was reported as a new compound. The absolute configuration of some compounds was established for the first time by electronic circular dichroism spectroscopy.

Comparison of UHPLC-ESI-MS and Hadamard transform atmospheric pressure ion mobility-ESI-MS for rapid profiling of isomeric flavonoids.

Hadamard transform atmospheric pressure ion mobility-MS and rapid UHPLC-MS methods were investigated for analysis of closely related isomeric flavonoids and their glycosides using a test set of seven standards. On a time scale of a few minutes, the flavonoid aglycones were all separated by ion mobility, but not by UHPLC. The glycosides were better resolved by IMS but not completely separated by both methods. The results suggest that IMS provides sufficient resolution for separation of isomeric polyphenols such as flavonoids in high-throughput metabolomics studies.

Statistical Correlations between HPLC Activity-Based Profiling Results and NMR/MS Microfraction Data to Deconvolute Bioactive Compounds in Mixtures

Recent approaches in natural product (NP) research are leading toward the discovery of bioactive chemical entities at the microgram level. In comparison to classical large scale bioassay-guided fractionation, the use of LC-MS metabolite profiling in combination with microfractionation for both bioactivity profiling and NMR analysis, allows the identification of bioactive compounds at a very early stage. In that context, this study aims to assess the potential of statistic correlation analysis to enable unambiguous identification of features related to bioactive compounds in mixtures, without the need for complete isolation. For that purpose, a mixture of NPs was microfractionated by rapid small-scale semi-preparative HPLC for proof-of-concept. UHPLC-ESI-TOFMS profiles, micro-flow CapNMR spectra and a cancer chemopreventive assay carried out on every microfraction were analysed by statistical correlations.

Pharmacognosy in the digital era: shifting to contextualized metabolomics

Humans have co-evolved alongside numerous other organisms, some having a profound effect on health and nutrition. As the earliest pharmaceutical subject, pharmacognosy has evolved into a meta-discipline devoted to natural biomedical agents and their functional properties. While the acquisition of expanding data volumes is ongoing, contextualization is lagging. Thus, we assert that the establishment of an integrated and open databases ecosystem will nurture the discipline. After proposing an epistemological framework of knowledge acquisition in pharmacognosy, this study focuses on recent computational and analytical approaches. It then elaborates on the flux of research data, where good practices could foster the implementation of more integrated systems, which will in turn help shaping the future of pharmacognosy and determine its constitutional societal relevance.

Dynamics of Metabolite Induction in Fungal Co-cultures by Metabolomics at Both Volatile and Non-volatile Levels

Fungal co-cultivation has emerged as a promising way for activating cryptic biosynthetic pathways and discovering novel antimicrobial metabolites. For the success of such studies, a key element remains the development of standardized co-cultivation methods compatible with high-throughput analytical procedures. To efficiently highlight induction processes, it is crucial to acquire a holistic view of intermicrobial communication at the molecular level. To tackle this issue, a strategy was developed based on the miniaturization of fungal cultures that allows for a concomitant survey of induction phenomena in volatile and non-volatile metabolomes. Fungi were directly grown in vials, and each sample was profiled by head space solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS), while the corresponding solid culture medium was analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS) after solvent extraction. This strategy was implemented for the screening of volatile and non-volatile metabolite inductions in an ecologically relevant fungal co-culture of Eutypa lata (Pers.) Tul. & C. Tul. (Diatrypaceae) and Botryosphaeria obtusa (Schwein.) Shoemaker (Botryosphaeriaceae), two wood-decaying fungi interacting in the context of esca disease of grapevine. For a comprehensive evaluation of the results, a multivariate data analysis combining Analysis of Variance and Partial Least Squares approaches, namely AMOPLS, was used to explore the complex LC-HRMS and GC-MS datasets and highlight dynamically induced compounds. A time-series study was carried out over 9 days, showing characteristic metabolite induction patterns in both volatile and non-volatile dimensions. Relevant links between the dynamics of expression of specific metabolite production were observed. In addition, the antifungal activity of 2-nonanone, a metabolite incrementally produced over time in the volatile fraction, was assessed against Eutypa lata and Botryosphaeria obtusa in an adapted bioassay set for volatile compounds. This compound has shown antifungal activity on both fungi and was found to be co-expressed with a known antifungal compound, O-methylmellein, induced in solid media. This strategy could help elucidate microbial inter- and intra-species cross-talk at various levels. Moreover, it supports the study of concerted defense/communication mechanisms for efficiently identifying original antimicrobials.

High Resolution Ion Mobility-ESI-MS for efficient profiling of natural products

The efficient analytical profiling of molecules in plant or fungal extracts represents a key element in natural product research. These complex extracts are typically characterized by highly chemodiverse secondary metabolites and by the presence of many multiple isomers. HPLC and UHPLC coupled with mass spectrometry are the most frequently used methods to analyze these types of samples but these techniques sometimes fail to resolve multiple isomers especially when used in a high throughput mode. In such cases, Ion Mobility Spectrometry (IMS) is an attractive alternative for the efficient separation of mixtures containing isomers, without increasing cycle time and with minimal additional experimental complexity [1,2]. In this study, the potential of High Resolution IMS-MS has been evaluated and compared to UHPLC-MS, for the analysis of closely related isomeric flavonoids and their glycosides. On a time scale of a few minutes, the flavonoid aglycones were all separated by ion mobility, but not by UHPLC. The glycosides were better resolved by IMS-MS, but not completely separated by both methods. The ion mobility resolving power was routinely > 150, indicating that the system provided sufficient resolution for a separation of isomeric natural products even in complex samples. Furthermore, HPLC-IMS-MS and UHPLC-MS were evaluated for the analysis of extracts from complex fungal co-cultures and mono-cultures. This study demonstrates that isomeric natural products can be better resolved by IMS-MS compared to UPLC-MS. Moreover, a significant number of compounds were detected by direct analysis of the fungal extracts using IMS-MS and were not detected by UHPLC-MS or MS only. These results suggest that high resolution IMS is well suited for the separation of isomeric compounds (even in high-throughput metabolomics studies) and appears as an attractive alternative to established UHPLC-MS methods. Keywords: Ion mobility-ESI-MS, separation of isomers, natural products, fingerprinting References: [1] H. H. Hill, W. F. Siems, R. H. St. Louis, D. G. McMinn. Anal Chem 1990; 62: 1201A [2] Z. Zhang, R. Knochenmuss, W. F. Siems, W. Liu, S. Graf, H. H. Hill. Anal Chem 2013; 86: 1661