MALDI-TOF Mass Spectrometry Imaging (MSI) for Distribution Analysis of Anthocyanins in Strawberry Fruits

Significance of the Topic

MALDI-TOF mass spectrometry imaging offers a powerful approach to map the spatial distribution of small-molecule compounds directly in biological tissues. In food science, this technique enables visualization of nutritionally and functionally important metabolites, such as anthocyanins in fruits. Understanding anthocyanin localization supports breeding of nutrient-rich varieties, quality control of produce, and development of functional food ingredients and nutraceuticals.

Objectives and Study Overview

This study aimed to identify major anthocyanin species in Tochiotome strawberries and visualize their distribution across fruit tissues, including skin, cortex, pith, and achenes. A preliminary extract-based profiling was performed to confirm compound identities before conducting high‐resolution imaging on thin tissue sections.

Methodology

Preparation of Crude Extracts:

• Freeze-drying and homogenization of ripe strawberries.
• Extraction in 50% methanol under shaking, centrifugation to collect supernatants.
• MALDI matrix mixing: 1 µL extract with 1 µL 40 mg/mL DHB in 70% methanol/0.1% formic acid on ITO slides.
• MS and MS/MS in positive-ion reflector mode (m/z 100–600) on Bruker ultrafleXtreme TOF/TOF.

Section Preparation for Imaging:

• Embedding fresh fruit in 2% carboxymethyl cellulose and flash‐freezing.
• Cryosectioning at 80 µm thickness (longitudinal, cross, and achene slices).
• Mounting on ITO slides, vacuum drying, storage at –80 °C.
• Matrix application via TM-Sprayer: 30 mg/mL DHB in 50% methanol/0.1% FA, 0.2 mL/min, 1200 mm/min, 81 passes, 85 °C.
• MSI data acquisition: 200 µm step size, medium laser diameter, positive-mode TOF/TOF.

Used Instrumentation

• Bruker ultrafleXtreme MALDI-TOF/TOF MS system
• HTX TM-Sprayer matrix deposition system
• Leica CM1860 cryostat
• flexAnalysis 3.4 and flexImaging 4.0 software (Bruker Daltonics)

Main Results and Discussion

Six anthocyanin glycosides were identified: pelargonidin-3-O-glucoside, cyanidin-3-O-glucoside, delphinidin-3-O-glycoside (first report in strawberry), pelargonidin-3-O-malonylglucoside, cyanidin-3-O-malonylglucoside, and pelargonidin-3-O-rutinoside. MS/MS neutral-loss patterns confirmed sugar attachments and aglycone forms.

MALDI-MSI revealed distinct spatial patterns:

• Pelargonidin derivatives localized in skin, cortex, and pith.
• Cyanidin and delphinidin derivatives confined to the skin layer.
• Achene sections showed strong signals for pelargonidin and cyanidin glycosides on the surface, with minimal delphinidin presence, correlating with reported high antioxidant capacity of achenes.

Benefits and Practical Applications

This imaging workflow delivers high‐resolution, label-free maps of bioactive compounds, facilitating:

• Screening of strawberry varieties for enhanced anthocyanin content.
• Monitoring of pigment development during ripening and under varying cultivation conditions.
• Optimization of extraction processes for nutraceutical and cosmetic industries.
• Assessment of compound absorption and distribution in food and topical applications.

Future Trends and Potential Applications

Advances in MALDI matrices and instrumentation promise higher spatial resolution and sensitivity. Integration with quantitative MSI and multivariate data analysis will enable precise concentration mapping. Extending this approach to diverse food matrices, plant breeding programs, and in vivo absorption studies will broaden applications in agriculture, nutrition, and pharmaceuticals.

Conclusion

MALDI-TOF MSI successfully identified and localized six anthocyanin species in Tochiotome strawberries, including delphinidin-3-O-glycoside for the first time. Critical sample preparation steps—CMC embedding, controlled matrix deposition, and elevated application temperature—ensured tissue integrity and analyte extraction. This technique offers a robust platform for spatially resolved metabolite profiling in food research and development.

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