MALDI-2 for enhanced in situ N-glycan analysis

Importance of the Topic

N-linked glycans are critical modulators of cell communication and are implicated in cancer, immune disorders, and viral infections, driving the need for spatially resolved glycan profiling
Advances in matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-MSI) combined with laser-induced postionization (MALDI-2) promise enhanced sensitivity and structural insight directly within tissue sections

Objectives and Overview of the Study

This work evaluates negative-ion mode MALDI-2 MSI on a timsTOF fleX trapped ion mobility quadrupole time-of-flight platform for in situ analysis of N-glycans in formalin-fixed paraffin-embedded human cerebellum
Key goals are to compare spatial glycan distributions versus traditional positive-ion MALDI-MSI methods and to demonstrate on-tissue tandem MS for structural elucidation of complex N-glycans

Methodology and Instrumentation

FFPE human cerebellum sections (5 μm) underwent dewaxing, rehydration, and homogeneous PNGase F application via automated sprayer with overnight incubation at 37 °C in humidified conditions
Nor-harmane served as MALDI matrix for negative-ion analyses (41 mM in 50 : 50 acetonitrile/water) and 2,5-dihydroxyacetophenone for positive-ion controls, both spiked with maltoheptaose as an internal standard
MALDI-2 MSI was performed on the timsTOF fleX MALDI-2 with N2 cooling at 3.0 mbar, MALDI laser 34 μJ, MALDI-2 laser 350 μJ, interlaser delay 30 μs, 50 shots per pixel, 1 kHz repetition rate, m/z 900–3000, 50 × 50 μm pixel size

Main Results and Discussion

Negative-ion MALDI-2 MSI yielded deprotonated N-glycan ions with spatial patterns closely matching positive-ion sodiated species, validating morphological fidelity across Purkinje, granular, molecular layers and white matter
A modest bias toward smaller glycans arose from in-source dissociation but did not alter overall tissue distributions
On-tissue low-energy CID MS/MS confirmed structures of 14 major N-glycans (e.g., H5N2), providing cross-ring fragments unique to negative-ion mode without off-tissue extraction
Correlation with mannosidase 1 alpha expression from human and mouse brain atlases supported localization in Purkinje and granular layers consistent with biosynthetic activity

Benefits and Practical Applications of the Method

Enhanced ion yields in negative-ion MALDI-2 enable high-quality tandem MS directly from tissue, preserving spatial context and delivering detailed glycan structures
This integrated imaging and structural approach accelerates discovery of glycan biomarkers, supports disease mechanism studies, and enhances QA/QC in pharmaceutical and clinical research

Future Trends and Applications

Coupling MALDI-2 MSI with ion mobility separation will improve isomer resolution and deepen insights into glycan heterogeneity
Advances in automated sample preparation and data analysis pipelines will facilitate high-throughput spatial glycomics in drug development and precision medicine
Extending MALDI-2 MSI to other posttranslational modifications and integrating with multiomic imaging will drive comprehensive tissue phenotyping at molecular resolution

Conclusion

Negative-ion mode MALDI-2 on a timsTOF fleX platform significantly boosts sensitivity for deprotonated N-glycans and enables direct on-tissue MS/MS structural elucidation
This method preserves spatial fidelity relative to positive-ion MALDI-MSI and offers a powerful complete solution for mass spectrometry–based glycomics research

References

• Soltwisch J Vens-Cappell S Wiegelmann M Müthing J Dreisewerd K Mass spectrometry imaging with laser-induced postionization Science 2015 348 211–215
• Heijs B Potthoff A Soltwisch J Dreisewerd K MALDI-2 for enhanced analysis of N-linked glycans by mass spectrometry imaging Anal Chem 2020 doi 10.1021/acs.analchem.0c02732
• Soltwisch J Heijs B Koch A Vens-Cappell S Höhndorf J Dreisewerd K MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument Anal Chem 2020 92 8697–8703
• Harvey DJ Negative ion mass spectrometry for the analysis of N-linked glycans Mass Spectrom Rev 2020 00 1–94
• Everest-Dass AV Briggs MT Kaur G Oehler MK Hoffmann P Packer NH N-glycan MALDI imaging mass spectrometry enables delineation of ovarian cancer tissues Mol Cell Proteomics 2016 15 3003–3016