Unlocking Nature's Secrets With Metabolome Informed Proteome Imaging

We developed and demonstrated a new metabolome-informed proteome imaging (MIPI) workflow for studying microscale microhabitats in complex ecosystems. Our workflow combines state-of-the art analytical instrumentation that generates spatial metabolome and proteome-rich data to build biological pathways.

In our roles as Pacific Northwest National Laboratory (PNNL) scientists, we led this multi-institutional study, which was published in Nature Chemical Biology.

Harnessing fungal communities for sustainable plant degradation and everyday products

A major research focus at PNNL is to understand how natural systems efficiently perform complex tasks, such as the breakdown of plants into high-value molecules. The leaf-cutter ant fungal garden ecosystem serves as a naturally evolved model for efficient plant biomass degradation. Characterizing the degradation processes mediated by the symbiotic fungus, Leucoagaricus gongylophorus (L. gongylophorus), is challenging due to the system's dynamic metabolisms and spatial complexity. In this study, we performed microscale imaging across sections of the Atta cephalotes fungal garden and used MIPI to map lignin degradation.

We focused on natural plant degradation because plants are vital to the global economy, creating products like fuels, medicines, insulation and food-safe packaging. Scientists seek cleaner, cheaper methods to break down tough plant materials, but current methods often leave behind waste, such as lignin. Natural fungal communities efficiently decompose these materials into usable nutrients. Understanding these processes at a molecular level can help us sustainably create everyday products.

Overcoming traditional limitations: MIPI unveils key biochemical pathways in fungal garden ecosystems

Traditional methods measure metabolites, enzymes and other molecules in bulk, providing average data that mask detailed information. MIPI offers detailed insights into biochemical pathways within complex biological matrices. Utilizing matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), MIPI visualizes metabolite locations and identifies microscale activity hotspots. These hotspots are then processed using microdroplet processing in one pot for trace samples (microPOTS), followed by sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analyses. This approach thoroughly examines the whereabouts, timing and molecular participants of biochemical reactions, providing a comprehensive view of intricate biological pathways.

Using our new imaging technique, MIPI, we examined 12 µm-thick sections of the leaf-cutter ant fungal garden ecosystem to map lignin degradation and visualize co-localized metabolites and proteins.

Untargeted MALDI-MSI analysis was used to look at molecular distributions across fungal garden sections. Using the METASPACE annotation platform and KEGG database, we identified metabolites and mapped distinct microscale lignin and primary metabolite microhabitats. These microscale microhabitats underwent detailed proteomic analysis using laser-capture microdissection and microPOTS followed by LC-MS/MS. Spatial metabolome and proteome data were then integrated.

The key findings of the paper were:

• Over 7,000 unique and taxon-specific peptides were identified from microscale samples. A diverse array of enzymes actively participating in the breakdown of lignocellulose and aromatic compounds were also revealed.
• A comprehensive view of metabolic pathways within microscale regions of this complex ecosystem was obtained from the integrated metabolome and proteome data.
• Critical metabolites and enzymes that drive essential biochemical reactions in plant degradation were identified using our MIPI method.
• Fungi were found to dominate and lignin breakdown pathways were identified. Our findings highlighted that fungi are the primary degraders of plant material in the system and provided detailed pathway-level resolution of lignin breakdown by the fungus.

MIPI reveals fungal dominance in plant material degradation and detailed pathways for lignin breakdown

Characterizing biochemical pathways in complex, heterogeneous samples has been challenging due to the limitations of current methods, which focus on bulk averages and obscure finer details. Our MIPI technique overcomes these limitations by visualizing individual molecular components, revealing specific biochemical reactions in plant degradation.

Using MIPI, we unveiled key metabolites and enzymes essential for these reactions within the leaf-cutter ant fungal garden ecosystem. We identified significant fungal involvement in breaking down plant material, particularly lignin. MIPI allowed us to observe co-localized metabolites and proteins, providing detailed pathway-level insights into lignin degradation by fungi.

Untargeted MALDI-MSI analysis highlighted heterogeneous spatial distributions of molecular features and identified distinct lignin and primary metabolite microhabitats. Detailed proteomic analysis using laser-capture microdissection and microPOTS, followed by LC-MS/MS, revealed thousands of unique and taxon-specific peptides. These results emphasized fungi's prominent role in lignocellulose degradation through various enzymes involved in carbohydrate and aromatic compound metabolism.

Through MIPI, we discovered that the fungus, L. gongylophorus, is the primary degrader of plant material and provided detailed pathways for lignin breakdown. These insights enhance our understanding of complex metabolic pathways, paving the way for advancements in both environmental research and biofuel and bioproduct development.

We now have a detailed microscale view of how plant degradation naturally occurs within this fungal system. To develop new methods for producing biofuels and bioproducts, we must start with the most basic components. By identifying which metabolites, enzymes and other chemicals initiate reactions at specific times and locations, we can replicate these processes in the lab. This knowledge can inform new strategies for creating better bioproducts and biofuels. We have successfully pinpointed the necessary molecules and biochemical reactions at a molecular level. With this natural blueprint, we can now apply the same techniques in the laboratory.

A current limitation of our MIPI workflow is the need for multiple adjacent tissue sections, as different modalities require specific sample slides. To address this, future efforts will focus on enabling MIPI to perform multi-omics profiling from a single tissue section while maintaining or even improving the sensitivity of all modalities.

Expanding horizons: Applying MIPI to future research and broader scientific endeavors

With our molecular-level understanding of these processes, we can now apply this method to a range of future experiments and broader scientific endeavors. We aim to study how fungal communities respond and protect themselves amid various disturbances, including those caused by extreme weather. Temperature fluctuations, shifts in precipitation patterns and increased exposure to pathogenic microbes can have devastating environmental impacts. Using this method, we can investigate how these communities respond to such stresses.

We are thrilled to have a tool that allows us to examine these processes in unprecedented detail, opening a whole new world of exploration.

Beyond environmental research, MIPI holds significant promise in clinical applications, offering valuable insights into cellular diversity and disease progression. Future research will leverage MIPI's capability to map biological systems intricately, investigating fungal responses to environmental stresses like extreme weather. This method not only deepens our understanding of these processes but also paves the way for new explorations and applications in both environmental and clinical settings.

Reference: Veličković M, Wu R, Gao Y, et al. Mapping microhabitats of lignocellulose decomposition by a microbial consortium. Nat Chem Biol. 2024;20(8):1033-1043. doi:10.1038/s41589-023-01536-7

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