Imaging venom peptides and proteins at high mass resolution, high lateral resolution and high speed using the timsTOF fleX

Significance of the topic

Understanding the spatial distribution of venom peptides and proteins within glandular tissue is critical for elucidating their biological functions, evolutionary origins and potential as therapeutic or agrochemical leads. MALDI mass spectrometry imaging (MSI) offers a label-free approach to map these biomolecules directly in situ, maintaining anatomical context and revealing compartmentalization that is lost in bulk analyses.

Objectives and study overview

This study aimed to demonstrate the capabilities of the timsTOF fleX platform for high-resolution MALDI-MSI of centipede venom glands. Key goals included achieving:

• High mass resolution and accuracy for peptides and small proteins (m/z 1 000–8 000 Da).
• High lateral resolution imaging (20 µm pixel size).
• High acquisition speed to generate rich spatial maps within practical timeframes.

Methodology

Centipede (Scolopendra morsitans) forcipules were fixed, dehydrated, cleared and paraffin-embedded before sectioning at 7 µm. Sections were mounted on ITO slides, deparaffinized and dried. Matrix (2,5-dihydroxybenzoic acid) was applied by sublimation under vacuum and recrystallized using a methanol/TFA spray protocol. Imaging was performed in QTOF mode over m/z 800–10 000 at 20 µm resolution, with 2 000 laser shots per pixel at 10 kHz. Data processing and visualization employed SCiLS Lab MVS, including root mean squared normalization and pLSA component analysis.

Used instrumentation

• timsTOF fleX in MALDI-MSI QTOF mode
• Bruker Peptide Calibration Standard II spiked with insulin for external calibration
• Bruker ImagePrep for matrix recrystallization
• SCiLS Lab MVS for data analysis and pLSA

Main results and discussion

The averaged mass spectrum revealed approximately 4 643 potential features, of which over 1 000 produced clear ion images. Key findings:

• Excellent mass resolution and accuracy (0–11 ppm) across m/z 1 000–6 500, demonstrated on a 6 001 Da venom peptide (U-SLPTX15-Sm1a).
• Dynamic range sufficient to visualize both high- and low-abundance components in a single dataset.
• Spatial resolution of 20 µm with acquisition speeds approaching 5 pixels per second for a dataset of 4 657 pixels.
• pLSA uncovered distinct spatial compartments within the venom gland, consistent with prior studies and suggesting functional heterogeneity in toxin storage.

Benefits and practical applications of the method

The timsTOF fleX MALDI-MSI workflow enables simultaneous detection of numerous venom peptides and proteins with preserved spatial context, surpassing probe-based methods and bulk proteomics in both throughput and anatomical resolution. This approach facilitates:

• Guided discovery of novel bioactive peptides.
• Integration with top-down and bottom-up proteomics for comprehensive toxin annotation.
• Insights into tissue-specific production and storage mechanisms of venom components.

Future trends and potential applications

Emerging developments are likely to include:

• Enhanced integration of ion mobility separation with MALDI-MSI for deeper molecular coverage.
• Advanced bioinformatics for automated feature annotation and correlation with genomic data.
• Application of high-resolution MSI to other venomous organisms and tissue types.
• Use of MSI-derived spatial maps to inform synthetic biology and rational toxin engineering.

Conclusion

The timsTOF fleX platform offers an unparalleled combination of mass accuracy, resolving power, spatial resolution and speed for MALDI imaging of venom peptides and small proteins. This capability paves the way for detailed spatially resolved venom research, bridging proteomic and genomic workflows, and accelerating both fundamental toxin biology studies and applied discovery efforts.

References

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