Interrogation of the Spatial Metabolome of Ginkgo biloba with high-resolution MALDI and LDI Mass Spectrometry Imaging

Significance of the Topic

Mapping the spatial distribution of metabolites within plant tissues provides critical insights into biosynthetic pathways and localized functions of bioactive compounds. Ginkgo biloba, a living fossil valued for its pharmacologically active flavonoids and terpenoids, presents a heterogeneous tissue structure that traditional homogenization-based analyses cannot resolve. High-resolution MALDI and LDI imaging open new avenues for in situ metabolomics, enabling label-free visualization of low-abundance molecules with cellular specificity.

Objectives and Study Overview

This study applied two complementary MALDI platforms to interrogate the spatial metabolome of Ginkgo biloba leaf cross sections. The primary goals were:

• Optimize matrix selection, coating method, mass range, and spatial resolution using ultrafleXtreme MALDI-TOF/TOF.
• Leverage solariX 7 T MRMS for high mass accuracy and specificity to map metabolites across tissue regions.
• Visualize and identify classes of compounds—flavonoid aglycones, biflavonoids, glycosides, biginkgosides, ginkgolides, ginkgolic acids and lipids—in situ.

Methodology and Sample Preparation

Fresh Ginkgo biloba leaves were embedded in gelatin and cryosectioned at 16 μm thickness. Sections were mounted onto ITO-coated slides and dehydrated under vacuum. Matrix deposition was performed using a custom aerosol sprayer (wet spraying) and by sublimation (dry spraying) for comparison. Tested matrices included:

• 2,5-Dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) in positive ion mode.
• 9-Aminoacridine (9-AA) in negative ion mode.

Optimal conditions (DHB with wet spraying) were selected based on signal intensity, metabolite coverage, and minimal analyte delocalization. Optical images guided region selection for mass spectrometry imaging.

Instrumental Setup

The following instruments were employed:

• Bruker ultrafleXtreme MALDI-TOF/TOF mass spectrometer for rapid screening, MS and MS/MS experiments.
• Bruker solariX 7 T MRMS with dual ESI/MALDI source and Smartbeam II laser for high-resolution imaging (m/z 150–2000) at 50 μm pixel size.
• Leica cryostat for tissue sectioning and Zeiss Axio M2 microscope for optical imaging.

Major Results and Discussion

Matrix optimization demonstrated that DHB in positive mode provided the richest spectral information, detecting bi-flavonoids, glycosides, and lipids. Wet spraying offered superior signal intensity and broader metabolite coverage compared to sublimation. High-resolution MRMS data enabled sub-ppm mass assignments and in situ MS/MS confirmation of compound classes. Key findings include:

• Spatial localization of flavonoid glycosides predominantly in the upper epidermis, with protonated, sodium and potassium adducts resolved by MRMS.
• Detection of flavonoid aglycone dimers (~m/z 1500) and deprotonated aglycones by LDI in negative mode.
• Visualization of biginkgosides with distinct distribution patterns reflecting chalcone synthase bioactivity in the upper epidermis.
• Mapping of phosphatidylcholines in mesophyll and secretory cavities, suggesting roles in intercellular signaling.

Benefits and Practical Applications

Label-free MALDI imaging overcomes limitations of tissue homogenization, revealing low-abundance metabolites and their organ-specific roles. This approach aids in:

• Understanding localized biosynthetic pathways and enzyme activity in plant tissues.
• Identifying target regions for extraction of pharmacologically active compounds.
• Informing breeding or engineering strategies to enhance desired metabolites.

Future Trends and Opportunities

Advancements in spatial resolution, multimodal imaging integration (optical, fluorescence), and machine learning for automated pattern recognition will further enhance plant metabolomics. Extending this methodology to diverse species and stress conditions promises to elucidate dynamic metabolic responses and support precision agriculture.

Conclusion

High-resolution MALDI and LDI imaging effectively mapped the heterogeneous metabolite landscape of Ginkgo biloba leaves, identifying key bioactive classes with cellular precision. These findings demonstrate the power of spatial metabolomics to deepen our understanding of plant biochemistry and pave the way for targeted exploitation of phytochemicals.

Reference

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