Coupling AP-SMALDI MS Imaging technology with a modified Orbitrap Hybrid mass spectrometer

Significance of the topic

Atmospheric-pressure scanning microprobe MALDI (AP-SMALDI) coupled with high-resolution Orbitrap mass analyzers enables spatially resolved molecular analysis at cellular to sub-cellular scales. This combination supports label-free localization of drugs, lipids and metabolites in tissues with high mass accuracy and high spatial resolution, improving confidence in molecular identification and enabling retrospective confirmation of compounds directly from tissue sections. The work summarized here demonstrates the AP-SMALDI5 AF ion source integrated with the new Thermo Scientific Orbitrap Excedion platform for advanced MS imaging (MSI) workflows.

Objectives and study overview

• Demonstrate the performance of AP-SMALDI5 AF coupled to the Orbitrap Excedion mass spectrometer for MS1 and MS2 imaging.
• Validate high spatial resolution imaging (down to 5 µm) on biological tissues and drug-dosed parasites.
• Compare ion funnel softness and resulting MS1 in-source fragmentation versus prior Orbitrap Exploris hardware.
• Showcase combined acquisition modes where MS1 and targeted MS2 (tMS2) data are collected in the same raw file and enable retrospective MS2 from preserved matrix-treated sections.

Materials and methods

• Samples: Healthy mouse brain (cerebellum), Fasciola hepatica (imatinib-dosed), Schistosoma mansoni (zotatifin-dosed). Tissue sections were cryosectioned at −20 °C and thaw-mounted on glass slides; S. mansoni was embedded in gelatine for sectioning.
• Matrix application: DHB and CHCA matrices were used. DHB was applied via the SMALDIPrep pneumatic sprayer (TransMIT) using tailored protocols; standard dried-droplet MALDI prep was used for some reference samples.
• Imaging parameters: Pixel sizes reported included 5 µm (MS1 imaging of mouse cerebellum and Schistosoma) and 20 µm (Fasciola), with positive-ion mode acquisitions covering roughly m/z 250–1000 and Orbitrap resolution settings up to 240k at m/z 200 where indicated. MS1 and tMS2 scans were interleaved or collected from adjacent pixels to generate combined MS1/MS2 images from a single raw file.
• Data processing: Ion images were generated using TransMIT’s MIRION software. Retrospective MS2 profiling was demonstrated using preserved tissue sections with DHB matrix still present.

Instrumentation

• Ion source: AP-SMALDI5 AF (TransMIT GmbH).
• Mass spectrometer: Thermo Scientific Orbitrap Excedion platform (Orbitrap Excedion Pro and Orbitrap Excedion MS variants reported).
• Additional ion source used for comparative stability tests: MassTech AP/MALDI UHR (used to confirm ion funnel softness independent of ion source model).

Main results and discussion

• Spatial resolution and localization: MS1 imaging resolved anatomical features in mouse cerebellum including a single layer of Purkinje cells at 5 µm pixel spacing, showing clear localization of distinct m/z channels corresponding to different tissue compartments.
• Drug localization and MS2 confirmation: Imatinib localization in Fasciola hepatica was mapped by MS1 and validated by tMS2 imaging showing precursor and characteristic fragment ions (e.g., m/z 217 and 393). Zotatifin (m/z 488.2183, formula C28H30N3O5) was localized in female Schistosoma mansoni and confirmed by a reference MS2 spectrum with characteristic fragments (e.g., m/z 168 and 58). Retrospective MS2 data taken months after initial acquisition (matrix preserved) successfully confirmed compounds.
• Softer ion funnel reduces in-source fragmentation: Comparative intensity-ratio experiments for labile amino acids (phenylalanine and isoleucine) and other test compounds indicate the Excedion platform’s ion funnel produces significantly less MS1 fragmentation than the Ion Funnel used with the Orbitrap Exploris 480. These findings were replicated using different AP/MALDI ion sources and matrices (DHB, CHCA), suggesting funnel softness is a robust instrument-inherent property rather than matrix-dependent under AP conditions.
• Data acquisition flexibility: The Excedion platform allowed combined MS1 and MS2 data acquisition within a single raw file (interleaved or adjacent-pixel tMS2), facilitating simultaneous imaging of distribution and structural confirmation without oversampling.
• Image quality and sensitivity: The combination of the AP-SMALDI ion source with the Excedion mass analyzer delivers high image sharpness and improved ion collection efficiency, yielding high-quality MSI datasets suitable for tissue histology correlation (matrix washed and H&E stained post-analysis).

Benefits and practical applications

• High-confidence molecular localization: High mass resolution and accurate MS2 confirmation reduce false positives from in-source fragments and support confident mapping of drugs and endogenous molecules in tissues.
• Cellular-scale imaging: 5 µm pixel spacing permits visualization of single-cell layers (e.g., Purkinje cells), enabling studies in neuroanatomy, pharmacodistribution, and cellular metabolomics.
• Retrospective confirmation: Preservation of matrix-treated sections allows MS2 confirmation after initial MS1 imaging, which is practical for compound ID workflows and follow-up analyses.
• Instrument compatibility and workflow integration: Demonstrated MALDI-readiness of the Orbitrap Excedion platform and compatibility with existing AP-SMALDI workflows and software (MIRION) supports adoption in research and QA/QC laboratories.

Future trends and applications

• Deeper integration of high-resolution MSI with histopathology and multi-omics, leveraging subcellular spatial resolution for correlative studies.
• Expanded use of retrospective MS2 and data-mining approaches to re-interrogate archived MSI datasets for new targets without re-acquisition.
• Optimization of ion funnel designs and atmospheric-pressure interfaces to further minimize in-source fragmentation across broader compound classes and ionization modes.
• Routine application in drug-distribution studies for parasitology, pharmacology and toxicology where localization and structural confirmation are critical.
• Development of standardized protocols for matrix application, preservation and automated retrospective MS2 workflows to increase reproducibility across labs.

Conclusion

The coupling of AP-SMALDI5 AF with the Thermo Scientific Orbitrap Excedion platform provides a robust MSI solution that combines high spatial resolution, high mass resolution and reduced in-source fragmentation. This platform supports MS1 imaging at cellular scales and reliable MS2 confirmation, including retrospective profiling from preserved sections. The softer ion funnel interface on the Excedion platform is a key contributor to reduced MS1 fragmentation and improved image interpretability, making the system well suited for biological and pharmaceutical MSI applications.

References

1. Spengler B, Roempp A, Guenther S, Schulz O, Hinz K-P, Hester AJ, Schinz C, Lotze C, Poetzl J-U, Strupat K. High Resolution in Mass and Space: AP-MALDI Imaging Technology for Orbitrap-based Instrumentation. ASMS 2012, Poster ID 241550.
2. Vegvari A, Strupat K, Dahlbäck M, Fehniger T, Marko-Varga G. Muscarinic Receptor Antagonist Target Disposition in Lung Disease Utilizing 10 µm Spatial Resolution of AP-SMALDI Tissue Imaging. ASMS 2013, Poster ID 236007.
3. Spengler B, Römpp A, Schäfer K.C. High Speed AP-MALDI Imaging at high spatial resolution (2D-Line mode). ASMS 2013, Poster ID 264750.
4. Spengler B, Dreisbach D, Schäfer K.-C., Morawietz C.M., Strupat K. MS and MS2 Imaging for compound confirmation: MS, tMS2, ddMS2 approaches for AP-SMALDI-MS Imaging with Orbitrap MS. ASMS 2023, Poster ID 314854.
5. Schäfer K.C., Liebschwager L., Strupat K., Spengler B. Comparison of instrumental synchronization modes in mass spectrometric imaging using Orbitrap mass analyzers. ASMS 2024, Poster ID 320451.
6. Thermo Fisher Scientific. Orbitrap Exploris 480 Software Manual, BRE0027667, Revision F, 2024.
7. AP-SMALDI5 AF Manual for Exploris and Excedion MS platforms, TransMIT GmbH, 2023/2026.

Acknowledgements: Financial support from the LOEWE Center for Novel Drug Targets against Poverty-Related and Neglected Tropical Infectious Diseases (DRUID) and DFG (SP314/23-1) is acknowledged. Some comparative ion source testing used MassTech AP/MALDI UHR. Authors include employees and collaborators of TransMIT GmbH and Thermo Fisher Scientific.