Mushroom Metabolomics: Mass Spectrometry and New Compounds

Mushroom Science

8 methods to find 3 compounds

A 2025 study used eight interdisciplinary analytical methods, including high-resolution mass spectrometry, NMR spectroscopy, and synthetic confirmation, to identify three previously unknown hericenone structures in lion's mane, one research team's illustration of how modern chemistry actually works.

Wang et al., Journal of Natural Products, 2025

1,353+

metabolite species identified in fungi by metabolomics platforms (Li et al., 2022)

Hundreds

distinct metabolites found in each of 6 common mushrooms by untargeted analysis (Hashimoto et al., 2023)

Varies widely

Chaga compound profiles differ dramatically between commercial products (Windsor et al., 2025)

Wang et al., Journal of Natural Products, 2025

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Most mushroom supplement labels discuss one metric: polysaccharide or beta-glucan content. Modern analytical chemistry, working through mass spectrometry and related instruments, has found hundreds of additional compounds in the same mushrooms that the standard polysaccharide test never detects. Some of those compounds are now the subject of active pharmaceutical research.

The 2025 discovery of three new hericenone structures in lion's mane is a useful entry point into how mass spectrometry mushroom compounds research actually works. The team that made that discovery used eight different analytical instruments before being confident in their results.^[1] This is not unusual for natural product chemistry. It reflects how different instruments see different aspects of the same molecule, and why no single method is sufficient.

This article explains what mass spectrometry does, why different MS instruments are used for different purposes in mushroom research, and what this science is revealing about compounds that were invisible to researchers a decade ago. For context on where this research sits in the broader picture, see Fruiting Body vs. Mycelium: The Great Mushroom Supplement Debate.

What Mass Spectrometry Actually Does to a Mushroom Sample

Mass spectrometry identifies molecules by measuring how heavy they are. The process starts by breaking the sample into individual molecules, then giving each molecule an electrical charge. Charged molecules move through a magnetic or electric field in the instrument, and heavier molecules separate from lighter ones based on how they travel through that field. A detector records which masses are present and at what abundance. The result is a spectrum, a graph of masses detected, that functions as a chemical fingerprint.

For mushroom research, the sample preparation matters as much as the instrument. A mushroom extract must first be separated into individual compounds before mass spectrometry can identify them. Liquid chromatography does this: the extract flows through a column packed with material that interacts differently with different molecules, causing compounds to arrive at the mass spectrometer at different times based on their chemical properties. Coupling liquid chromatography to mass spectrometry creates the LC-MS method used in most modern mushroom metabolomics studies.

The second major technique in mushroom chemistry is MALDI-TOF, which stands for matrix-assisted laser desorption/ionization time-of-flight. MALDI works differently from LC-MS: the sample is mixed with a matrix material and spotted onto a metal plate. A laser vaporizes and ionizes the compounds directly from the surface. The "time-of-flight" part measures how long each ionized molecule takes to travel to a detector; heavier molecules travel more slowly. MALDI-TOF is particularly useful for analyzing large molecules like polysaccharides, which would fragment under other ionization conditions.

What mass spectrometry reveals that earlier methods missed:

• Targeted analysis measures specific known compounds you are looking for
• Untargeted metabolomics measures everything simultaneously, including compounds you didn't know to look for
• High-resolution MS determines exact molecular formulas, not just molecular weight
• Tandem MS (MS/MS) fragments molecules and measures the fragments to reveal structural details

A 2022 review by Li and colleagues mapped what modern metabolomics approaches have identified in fungi: 1,353 or more metabolite species in studied fungal samples simultaneously, including sugars, glycosides, amino acids, phenols, flavonoids, and terpenes as distinct compound groups.^[10] Standard supplement testing measures a fraction of this chemical space.

The 2025 Lion's Mane Discovery: What It Actually Takes to Find a New Compound

The 2025 study by Wang and colleagues on lion's mane is worth following in methodological detail because it shows what "we found a new compound" actually means in practice.^[1]

The research team was working with lion's mane fruiting bodies, targeting hericenones, the aromatic compounds in lion's mane associated with nerve growth factor stimulation. They suspected there were more hericenone variants than had been characterized. Finding and confirming them required eight separate analytical methods working in sequence.

High-resolution mass spectrometry (HR-MS) provided the first clue: peaks in the spectrum that didn't match any known hericenone mass. But mass alone doesn't determine structure. The team then used 2D NMR spectroscopy (two-dimensional nuclear magnetic resonance) to map the three-dimensional arrangement of atoms in the candidate molecules. NMR works by placing the sample in a strong magnetic field and measuring how atomic nuclei respond to radio pulses; different atoms in different structural positions respond at different frequencies, allowing the spatial arrangement to be mapped. The 2D variants of NMR provide additional information about which atoms are connected to or near each other.

After NMR, the researchers used circular dichroism (CD) spectroscopy to determine which mirror-image form (enantiomer) of the molecule they had found. Many biological compounds come in two mirror-image forms that interact differently with receptors. CD spectroscopy distinguishes them by measuring how the molecule interacts with circularly polarized light.

Finally, the team confirmed all three new hericenone structures by total synthesis: they built the molecules from scratch in the laboratory using known chemical reactions. If the synthetic version showed identical spectral data to the natural extract version, the structure was confirmed.

Eight methods for three compounds. This is the current standard for confident natural product identification, and it is why the pace of new mushroom compound discovery is slow relative to what metabolomics implies exists.

Two Types of Mass Spec That Serve Different Research Purposes: LC-MS and MALDI-TOF

The two dominant mass spectrometry approaches in mushroom research answer different questions. Understanding which does which explains why researchers combine them rather than choosing one.

LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) excels at identifying small and medium-sized molecules in complex mixtures. Because liquid chromatography separates compounds before they reach the mass spectrometer, LC-MS can simultaneously characterize dozens or hundreds of individual compounds in a single mushroom extract. Its tandem capability (fragmenting molecules in a second MS step) provides additional structural information.

A 2025 study by Randeni and colleagues used LC-MS to characterize compounds in Chaga mushroom, then fed those identifications into a network pharmacology and molecular docking pipeline.^[2] Network pharmacology maps which identified compounds are likely to interact with which molecular targets in the body, based on known compound-target databases. Molecular docking simulates how well a compound physically fits into a protein binding site, like a key into a lock. The result: a pathway from "here are the molecules in Chaga" to "here is how they likely interact with anti-diabetic biological targets." This integrative chemistry-to-mechanism pipeline represents the current frontier of mushroom compound research.

MALDI-TOF works differently and is particularly suited to large molecules. A 2016 study by López-García and colleagues demonstrated that MALDI-TOF could produce distinct polysaccharide mass fingerprints for different mushroom species in commercial supplements, enabling species authentication.^[4] The instrument essentially reads a unique polysaccharide signature from each species, similar to how a barcode encodes identity. Two different mushroom species produce different MALDI-TOF fingerprints even when their polysaccharide content looks similar by older assay methods.

For supplement quality applications, MALDI-TOF authentication addresses one of the most persistent problems in the mushroom supplement market: products that are labeled as one species but contain another, or contain mixtures not reflected on the label.

When the Analytical Method Choice Changes the Answer

A critical and underappreciated finding in mushroom analytical chemistry is that different measurement methods give different results for the same compounds in the same sample. This is not measurement error; it is a consequence of how each method works.

A 2021 comparative study by Tsiantas and colleagues measured ergothioneine and lovastatin (a cholesterol-relevant polyketide naturally present in some mushrooms) across multiple mushroom species using three different analytical approaches: HPLC, LC-MS/MS, and NMR spectroscopy.^[5] The results varied depending on which method was used, with different methods showing different sensitivity thresholds and different abilities to distinguish closely related compounds.

The practical consequence: when different studies report different concentrations of the same compound in the same mushroom species, the discrepancy may be entirely attributable to analytical method differences rather than genuine biological variation. A company measuring ergothioneine by HPLC and another measuring it by LC-MS may report different numbers for the same starting material.

This has direct relevance for supplement labels that report compound quantities. The method used to generate that number matters as much as the number itself, and labels rarely disclose measurement methodology. The variability in compound profiles between commercial mushroom products is explored in Do Fruiting Bodies Have More Bioactive Compounds Than Mycelium? from the perspective of source material, and in How Growing Substrate Affects Mushroom Bioactive Content from the cultivation angle.

What Untargeted Metabolomics Has Found in Familiar Mushrooms

The most striking finding from applying untargeted metabolomics to familiar edible mushrooms is not about exotic medicinal species. It is about button mushrooms, shiitakes, and chanterelles.

A 2023 untargeted metabolomics study by Hashimoto and colleagues profiled six common mushroom species: white button, crimini, portabella, lion's mane, maitake, and shiitake.^[7] Each species showed a unique chemical fingerprint consisting of hundreds of distinct metabolites. The enriched compound classes were not primarily polysaccharides: terpenoids, fatty acids, glycerophospholipids (a class of molecules that form cell membranes), and sphingolipids (another membrane lipid class with signaling roles) dominated the species-specific profiles.

The chanterelle is similarly surprising on analysis. A 2023 metabolomics study by Režić Mužinić and colleagues profiled aqueous extracts of golden chanterelle and found a complex mixture of phenolics, flavonoids, terpenoids, and organic acids, all present in a mushroom typically evaluated only for its culinary value and nutritional content.^[9]

Metabolomics also reveals how dynamically a mushroom's chemistry changes over time. A 2019 study by Oh and colleagues tracked metabolite profiles in Cordyceps militaris as the fruiting bodies aged and found that cordycepin, the primary bioactive compound associated with anti-inflammatory effects, increases dramatically in concentration as the mushroom matures.^[8] Harvesting Cordyceps at different stages of maturity produces products with measurably different cordycepin content. This was invisible to older analytical approaches that measured a single compound at a single time point; metabolomics tracks the full profile in real time. For deeper coverage of cordycepin specifically, see Cordycepin (3'-Deoxyadenosine): The Multi-Target Compound Behind Cordyceps' Effects.

The Consumer Stakes: Why Mass Spec Data Changes How You Evaluate Supplements

The science of mass spectrometry mushroom compounds research has a direct consumer implication: the chemical profile of a mushroom product depends on which species was used, how it was grown, when it was harvested, how it was processed, and how the final product was analyzed. Mass spectrometry is the only method that can verify all of these factors simultaneously.

A 2021 metabolomics study by Nam and colleagues compared shiitake mushrooms grown on traditional logs versus on modern sawdust substrate.^[6] Untargeted metabolomics revealed that substrate choice affects dozens of secondary metabolite classes simultaneously, not just a single compound like beta-glucan. Log-grown shiitake had significantly different metabolic profiles from sawdust-grown shiitake across multiple compound families.

The same principle extends to commercial supplement products. A 2025 comparative study by Windsor and colleagues applied multiple complementary analytical techniques to commercial Chaga products.^[3] The study found enormous variation in compound profiles between products. Products with equivalent label claims showed different compound content, different ratios of compound classes, and in some cases, profiles inconsistent with authentic Chaga. Mass spectrometry made these differences visible where label-level polysaccharide testing alone would not.

The broader implications of this chemistry are covered in Mushroom Metabolomics: Mapping Every Compound in a Mushroom, Secondary Metabolites in Mushrooms: The Compounds Beyond Polysaccharides, and the AI-accelerated future of compound discovery in AI-Driven Mushroom Compound Discovery: The Future of Mycochemistry. For what happens when chemistry advances translate into the broader field, see How Modern Chemistry Is Revealing New Mushroom Compounds.

The Bottom Line

Mass spectrometry identifies mushroom compounds by measuring molecular mass, separating hundreds of compounds simultaneously in a single extract, and providing structural information that allows new compound identification. The 2025 discovery of three new hericenone structures in lion's mane required eight analytical methods, including high-resolution MS, NMR, circular dichroism, and synthetic confirmation. Different MS instruments serve different purposes: LC-MS/MS works best for small molecule metabolomics; MALDI-TOF for polysaccharide fingerprinting and species authentication. Untargeted metabolomics has revealed hundreds of previously uncharacterized compounds in common edible mushrooms. Commercial supplement products for the same species show widely different compound profiles under modern analytical methods, illustrating both the power and the gap between laboratory chemistry and market claims.

Frequently Asked Questions

How does mass spectrometry work for mushrooms?

Mass spectrometry identifies compounds in mushroom extracts by measuring molecular mass. A mushroom extract is separated into individual compounds by liquid chromatography, then each compound passes through a mass spectrometer that ionizes (electrically charges) the molecules and measures how their mass-to-charge ratio compares as they travel through an electric or magnetic field. The result is a mass spectrum, a fingerprint of all masses detected, that can be matched against databases of known compounds or used to identify new ones. LC-MS/MS is the most common approach for broad metabolite profiling.

What is LC-MS/MS in mushroom research?

LC-MS/MS stands for liquid chromatography tandem mass spectrometry. The liquid chromatography step separates a complex mushroom extract into individual compounds by flowing it through a column that interacts differently with different molecules; more-retained compounds emerge later, creating a time-separated stream. The tandem mass spectrometry step first records the mass of each compound, then fragments it and records the fragment masses, providing structural information beyond molecular weight alone. This approach can identify and quantify hundreds of compounds simultaneously in a single run.

What new mushroom compounds have been found using mass spectrometry?

A 2025 study used high-resolution mass spectrometry alongside seven other analytical methods to identify three previously unknown hericenone structures in lion's mane (Hericium erinaceus). A 2023 untargeted metabolomics study found hundreds of distinct metabolites in six common mushroom species, including terpenoids, glycerophospholipids, and sphingolipids not typically captured by standard polysaccharide testing. Metabolomics has also tracked how Cordyceps militaris cordycepin content changes with fruiting body age and how shiitake's metabolite profile shifts based on whether it was grown on logs or sawdust.

What is the difference between MALDI-TOF and LC-MS for mushrooms?

MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) uses a laser to vaporize compounds from a prepared surface, then measures how long each ionized molecule takes to reach a detector. It excels at analyzing large molecules like polysaccharides and can produce species-specific polysaccharide fingerprints for supplement authentication. LC-MS couples liquid chromatography separation with mass spectrometry detection and is better suited to identifying and quantifying many small to medium-sized compounds simultaneously. The two methods provide complementary information; comprehensive mushroom chemical characterization typically uses both.

How do scientists identify unknown compounds in mushrooms?

Identifying an unknown mushroom compound requires multiple instruments working in sequence. High-resolution mass spectrometry provides the molecular formula. 2D NMR spectroscopy (nuclear magnetic resonance, a technique that maps how atoms are connected using magnetic fields) determines three-dimensional structure. Circular dichroism spectroscopy distinguishes between mirror-image forms of the molecule. Total synthesis (building the compound from scratch using known chemical reactions) provides the final structural confirmation by comparing the synthetic and natural versions. The 2025 lion's mane hericenone study used 8 instruments total to confirm 3 new structures, reflecting the current standard for natural product identification.

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