Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

Antibacterial Synnepyrroles from Human-Associated Nocardiopsis sp. Show Protonophore Activity and Disrupt the Bacterial Cytoplasmic Membrane.

Actinobacteria have traditionally been an important source of bioactive natural products, although many genera remain poorly explored. Here, we report a group of distinctive pyrrole-containing natural products, named synnepyrroles, from Nocardiopsis synnemataformans. Detailed structural characterization by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy combined with isotope-labeling experiments revealed their molecular structures and biosynthetic precursors acetate, propionate, aspartate, and (for branched analogues) valine. The biosynthetic data points toward an unusual pathway for pyrrole formation via condensation of aspartate with diverse fatty acids that give rise to a unique pyrrole-3,4-dicarboxylate core and variable linear or terminally branched alkyl side chains. In addition, the bioactivity and mode of action of synnepyrrole A were characterized in Bacillus subtilis. Orienting assessment of the phenotype of synnepyrrole A-treated bacteria by high-resolution microscopy suggested the cytoplasmic membrane as the target structure. Further characterization of the membrane effects demonstrated dissipation of the membrane potential and intracellular acidification indicative of protonophore activity. At slightly higher concentrations, synnepyrrole A compromised the barrier function of the cytoplasmic membrane, allowing the passage of otherwise membrane-impermeable dye molecules.

Efficient bio-degradation of food waste through improving the microbial community compositions by newly isolated Bacillus strains.

This study aims to screen high-degradability strains and develop a novel microbial agent for efficient food waste degradation. The effects of the novel microbial agent on organic matter degradation, enzyme activity, and bacterial succession during the in-situ reduction of food waste were evaluated and compared with other two microbial agents previously developed. Results showed that the novel agent containing four Bacillus strains received maximum organic degradation rates, volatile solid removal (46.91%) and total mass reduction (76.16%). Pyrosequencing analysis revealed that there was a significant difference in the microbial community structure of the matrix among the three biodegradation systems, and the novel agent greatly improved the stability of in-situ reduction process that Bacillus was the dominant genus (>98%) since day 4. These results indicated that the inoculant containing only Bacillus was more stable and cost-effective in FW in-situ reduction.