RESUMO
The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.
Assuntos
Lignina , Consórcios Microbianos , Lignina/metabolismo , Consórcios Microbianos/fisiologia , Animais , Formigas/metabolismo , Formigas/microbiologia , Ecossistema , Proteômica/métodos , Proteoma/metabolismo , SimbioseRESUMO
Understanding fungal lipid biology and metabolism is critical for antifungal target discovery as lipids play central roles in cellular processes. Nuances in lipid structural differences can significantly impact their functions, making it necessary to characterize lipids in detail to understand their roles in these complex systems. In particular, lipid double bond (DB) locations are an important component of lipid structure that can only be determined using a few specialized analytical techniques. Ozone-induced dissociation mass spectrometry (OzID-MS) is one such technique that uses ozone to break lipid DBs, producing pairs of characteristic fragments that allow the determination of DB positions. In this work, we apply OzID-MS and LipidOz software to analyze the complex lipids of Saccharomyces cerevisiae yeast strains transformed with different fatty acid desaturases from Histoplasma capsulatum to determine the specific unsaturated lipids produced. The automated data analysis in LipidOz made the determination of DB positions from this large dataset more practical, but manual verification for all targets was still time-consuming. The DL model reduces manual involvement in data analysis, but since it was trained using mammalian lipid extracts, the prediction accuracy on yeast-derived data was reduced. We addressed both shortcomings by retraining the DL model to act as a pre-filter to prioritize targets for automated analysis, providing confident manually verified results but requiring less computational time and manual effort. Our workflow resulted in the determination of detailed DB positions and enzymatic specificity.
Assuntos
Aprendizado Profundo , Ozônio , Saccharomyces cerevisiae , Fluxo de Trabalho , Saccharomyces cerevisiae/química , Ozônio/química , Histoplasma/química , Histoplasma/metabolismo , Espectrometria de Massas/métodos , Software , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos/química , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Lipídeos/químicaRESUMO
Mass spectrometry is broadly employed to study complex molecular mechanisms in various biological and environmental fields, enabling 'omics' research such as proteomics, metabolomics, and lipidomics. As study cohorts grow larger and more complex with dozens to hundreds of samples, the need for robust quality control (QC) measures through automated software tools becomes paramount to ensure the integrity, high quality, and validity of scientific conclusions from downstream analyses and minimize the waste of resources. Since existing QC tools are mostly dedicated to proteomics, automated solutions supporting metabolomics are needed. To address this need, we developed the software PeakQC, a tool for automated QC of MS data that is independent of omics molecular types (i.e., omics-agnostic). It allows automated extraction and inspection of peak metrics of precursor ions (e.g., errors in mass, retention time, arrival time) and supports various instrumentations and acquisition types, from infusion experiments or using liquid chromatography and/or ion mobility spectrometry front-end separations and with/without fragmentation spectra from data-dependent or independent acquisition analyses. Diagnostic plots for fragmentation spectra are also generated. Here, we describe and illustrate PeakQC's functionalities using different representative data sets, demonstrating its utility as a valuable tool for enhancing the quality and reliability of omics mass spectrometry analyses.