Large scale MALDI-TOF imaging of metabolites from filamentous fungi

Significance of the Topic

Imaging mass spectrometry by MALDI-TOF has become a vital tool for mapping the spatial distribution of metabolites in biological samples with minimal preparation and high spatial resolution. In microbial research, this technique enables direct visualization of metabolic interactions within intact colonies, supporting studies of microbial communities, microbiome dynamics and interspecies exchange of small molecules. Filamentous fungi present unique challenges due to deep-penetrating hyphae, nonuniform matrix adherence and sample flaking under high vacuum. Addressing these issues expands the applicability of MALDI imaging to fungal metabolomics and microbial co-culture systems where spatial context is crucial.

Study Objectives and Overview

This work aimed to develop a reproducible method for large-scale MALDI-TOF imaging of filamentous fungi on a 384-spot ground steel plate. By optimizing sample preparation—particularly matrix application and agar section adhesion—the study sought to overcome limitations encountered on conventional 96-spot plates. Key goals included:

1. Enhancing consistency between runs through improved matrix deposition.
2. Maintaining colony morphology and spatial separation of secreted versus colony-associated metabolites.
3. Enabling simultaneous imaging of multiple conditions and time points in a single acquisition.

Methodology and Instrumentation

Matrix application was performed using an HTX TM Sprayer to deposit a 1:1 CHCA:DHB solution at 5 mg/mL in 90% acetonitrile with 0.1% TFA. Parameters included nine crisscross passes at 0.2 mL/min, 30 °C, 40 mm nozzle height and a calculated density of 2.7 μg/mm2. Pre-coated plates served as an adhesive layer for agar sections excised from Petri dishes. Fungal colonies were gently flattened with a sterile swab to remove fluffy hyphae and prevent spore ingress during analysis. Samples were desiccated at 30 °C until fully dry, followed by a second identical matrix spray to ensure uniform coverage. The ground steel plate choice increased surface roughness and adhesion compared to polished targets, crucial for high-vacuum stability.

Main Results and Discussion

The 384-spot format provided four times the analysis area of standard 96-spot plates, enabling 18 conditions and over 13 000 spectra in a single run at 500 μm resolution. Time-course imaging revealed dynamic metabolite distributions: an m/z 309.6 signal emerged only at later growth stages, while m/z 598.6 initially localized near colonies before diffusing into agar. Co-culture experiments distinguished metabolite sources by including individual microbial controls. SCiLS statistical segmentation highlighted unique metabolic regions particularly prominent in day-7 fungal growth. Principal component analysis of segmented regions showed that metabolite profiles cluster more strongly by microbial composition than by growth duration, with notable outliers corresponding to unique secondary metabolites from specific yeast strains.

Benefits and Practical Applications

• Simultaneous comparison of multiple strains, conditions and time points accelerates screening of microbial interactions and metabolic exchange.
• Enhanced sample adhesion prevents flaking, ensuring robust data acquisition under high vacuum.
• Ground steel plates combined with TM Sprayer application provide uniform matrix coverage over large areas.
• Large data sets facilitate advanced statistical analyses, increasing confidence in metabolomic interpretations.

Future Trends and Potential Applications

Advances in MALDI imaging will likely focus on further automation of sample preparation, integration of additional media types and expansion to more complex multi-species communities. Development of complementary matrices that target a wider range of metabolite chemistries and refinements in spatial resolution will enhance detection sensitivity. Coupling MALDI imaging with orthogonal techniques such as ion mobility or tandem MS workflows may provide deeper structural insight. As statistical tools evolve, real-time data processing and machine-learning-driven feature extraction will drive high-throughput microbial metabolomics.

Conclusion

This optimized protocol for large-scale MALDI-TOF imaging on 384-spot ground steel plates delivers consistent, high-resolution molecular maps of filamentous fungal colonies and microbial co-cultures. The improved matrix application and sample handling preserve colony morphology and maximize data quality, enabling comprehensive comparisons across multiple conditions in a single analysis. This approach streamlines fungal metabolomics workflows and lays the groundwork for more complex microbiome imaging studies.

Instrumentation

The key instrumentation included:

• Autoflex Speed MALDI-TOF mass spectrometer.
• HTX Technologies TM Sprayer for controlled matrix deposition.
• Bruker 384-spot ground steel MALDI target plates.

References

• Huang M-Z, Cheng S-C, Jhang S-S et al. Ambient molecular imaging of dry fungus surface by electrospray laser desorption ionization mass spectrometry. Int J Mass Spectrom. 325-327:172-181 (2012).
• Moree WJ, Phelan VV, Wu CH et al. Interkingdom metabolic transformations captured by microbial imaging mass spectrometry. Proc Natl Acad Sci USA. 109(34):13811-13816 (2012).
• Yang JY, Phelan VV, Simkovsky R et al. Primer on agar-based microbial imaging mass spectrometry. J Bacteriol. 194(22):6023-6028 (2012).