Untargeted and Targeted LC-MS/MS Based Metabolomics Study on In Vitro Culture of Phaeoacremonium Species

Grapevine (Vitis vinifera L.) can be affected by many different biotic agents, including tracheomycotic fungi such as Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are the main causal agent of Esca and Petri diseases. Both fungi produce phytotoxic naphthalenone polyketides, namely scytalone and isosclerone, that are related to symptom development. The main objective of this study was to investigate the secondary metabolites produced by three Phaeoacremonium species and to assess their phytotoxicity by in vitro bioassay. To this aim, untargeted and targeted LC-MS/MS-based metabolomics were performed. High resolution mass spectrometer UHPLC-Orbitrap was used for the untargeted profiling and dereplication of secondary metabolites. A sensitive multi reaction monitoring (MRM) method for the absolute quantification of scytalone and isosclerone was developed on a UPLC-QTrap. Different isolates of P. italicum, P. alvesii and P. rubrigenum were grown in vitro and the culture filtrates and organic extracts were assayed for phytotoxicity. The toxic effects varied within and among fungal isolates. Isosclerone and scytalone were dereplicated by matching retention times and HRMS and MS/MS data with pure standards. The amount of scytalone and isosclerone differed within and among fungal species. To our best knowledge, this is the first study that applies an approach of LC-MS/MS-based metabolomics to investigate differences in the metabolic composition of organic extracts of Phaeoacremonium species culture filtrates.

On the use of a 2D-carbon microfiber fractionation system to improve flow-injection QTOF-HRMS analysis in complex matrices: the case of Abelmoschus manihot flower extracts

A two-dimensional microscale carbon fiber/active carbon fiber system combined with a quadrupole time of flight high-resolution mass spectrometer (2DCFs-QTOF-HRMS) system is proposed to rapidly putative identify polar, medium polar and...

A Generic LC-HRMS Screening Method for Marine and Freshwater Phycotoxins in Fish, Shellfish, Water, and Supplements

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

A SISCAPA-based approach for detection of SARS-CoV-2 viral antigens from clinical samples

SARS-CoV-2, a novel human coronavirus, has created a global disease burden infecting > 100 million humans in just over a year. RT-PCR is currently the predominant method of diagnosing this viral infection although a variety of tests to detect viral antigens have also been developed. In this study, we adopted a SISCAPA-based enrichment approach using anti-peptide antibodies generated against peptides from the nucleocapsid protein of SARS-CoV-2. We developed a targeted workflow in which nasopharyngeal swab samples were digested followed by enrichment of viral peptides using the anti-peptide antibodies and targeted parallel reaction monitoring (PRM) analysis using a high-resolution mass spectrometer. This workflow was applied to 41 RT-PCR-confirmed clinical SARS-CoV-2 positive nasopharyngeal swab samples and 30 negative samples. The workflow employed was highly specific as none of the target peptides were detected in negative samples. Further, the detected peptides showed a positive correlation with the viral loads as measured by RT-PCR Ct values. The SISCAPA-based platform described in the current study can serve as an alternative method for SARS-CoV-2 viral detection and can also be applied for detecting other microbial pathogens directly from clinical samples.

Complexes with Atomic Gold Ions: Efficient Bis-Ligand Formation

Complexes of atomic gold with a variety of ligands have been formed by passing helium nanodroplets (HNDs) through two pickup cells containing gold vapor and the vapor of another dopant, namely a rare gas, a diatomic molecule (H2, N2, O2, I2, P2), or various polyatomic molecules (H2O, CO2, SF6, C6H6, adamantane, imidazole, dicyclopentadiene, and fullerene). The doped HNDs were irradiated by electrons; ensuing cations were identified in a high-resolution mass spectrometer. Anions were detected for benzene, dicyclopentadiene, and fullerene. For most ligands L, the abundance distribution of AuLn+ versus size n displays a remarkable enhancement at n = 2. The propensity towards bis-ligand formation is attributed to the formation of covalent bonds in Au+L2 which adopt a dumbbell structure, L-Au+-L, as previously found for L = Xe and C60. Another interesting observation is the effect of gold on the degree of ionization-induced intramolecular fragmentation. For most systems gold enhances the fragmentation, i.e., intramolecular fragmentation in AuLn+ is larger than in pure Ln+. Hydrogen, on the other hand, behaves differently, as intramolecular fragmentation in Au(H2)n+ is weaker than in pure (H2)n+ by an order of magnitude.

Overexpression of Global Regulator Talae1 Leads to the Discovery of New Antifungal Polyketides From Endophytic Fungus Trichoderma afroharzianum

Transcription regulation caused by global regulators exerts important effects on fungal secondary metabolism. By overexpression of the global regulator Talae1 in a Ficus elastica-associated fungus Trichoderma afroharzianum, two structurally new polyketides (1 and 2) that were newly produced in the transformant were isolated and identified. Their structures, including the absolute configurations, were elucidated through a combination of high resolution mass spectrometer (HRMS), NMR, and electronic circular dichroism (ECD) calculations. The growth inhibitory activities of compounds 1 and 2 were evaluated against four bacteria and six plant-pathogenic fungi. Compound 1 showed the highest antifungal activity against Botrytis cinerea and Fusarium oxysporum f. sp. nicotianae with MIC of 8 μg/ml. To the best of our knowledge, this is the first study to report on the application of the global regulator in T. afroharzianum to activate the biosynthesis of bioactive secondary metabolites.