Advancing spatial lipidomics: high spatial resolution mass spectral imaging (MSI) using the MassTech AP-MALDI UHR ion source with the Orbitrap Exploris mass spectrometer

Significance of the topic

Mass spectrometry imaging (MSI) with atmospheric-pressure MALDI (AP-MALDI) bridges molecular specificity and spatial resolution to map lipids directly in tissue sections without extraction. High spatial-resolution AP-MALDI coupled to high-resolution accurate-mass (HRAM) Orbitrap analysis enables confident identification and localization of lipid species in small and delicate samples such as reconstructed human epidermis (RHE). This capability is particularly relevant for dermatological research, inflammatory pathway studies, and development/assessment of topical personal-care products where cellular- and layer-specific lipid changes are critical.

Goals and overview of the study

This application note demonstrates the performance and practical utility of the MassTech AP-MALDI UHR ion source combined with a Thermo Scientific Orbitrap Exploris mass spectrometer for high-spatial-resolution lipid MSI. The study focuses on: mapping and annotating lipids across RHE layers at fine spatial scales (down to 5 μm step size), comparing lipid distributions under solar-simulated UV radiation with and without a sun-filter formulation, and establishing a workflow that integrates MSI with complementary LC-MS lipidomics using the same instrument platform.

Methodology

The experimental workflow encompassed RHE sample preparation, sectioning, matrix deposition, AP-MALDI MSI acquisition, and data processing. Key procedural points include:

• RHE exposure: models were either left untreated, irradiated with solar-simulated radiation (SSR) at 16.5 J/cm2 (2× MED), or pre-treated with a sun-filter formulation prior to SSR and incubated 24 h post-irradiation.
• Embedding and sectioning: RHE specimens embedded in a gelatin (10%) + carboxymethylcellulose (2.5%) mixture, flash-frozen, and cryo-sectioned at 6 μm using a Cryo-Ultramicrotome at −20 °C. Sections were thaw-mounted onto APTES-coated stainless-steel plates.
• Matrix application: either HCCA (α-cyano-4-hydroxycinnamic acid) or 1,5-diaminonaphthalene (DAN) applied by automated spraying (SunCollect MALDI-Sprayer) with parameters tuned for high-resolution lipid imaging.
• MSI acquisition: Constant Speed Raster mode with 5 μm step size, laser at 400 Hz and low power (3%), ion injection time 490 ms, mass range m/z 205–2000, Orbitrap resolution set to 240,000 at m/z 200. Automatic gain control (AGC) disabled to ensure constant injection time across pixels. Data acquired in both positive and negative ion modes with EASY-IC lock mass for scan-to-scan correction.
• Data processing: raw files converted to imzML and analyzed with LipostarMSI for image visualization, segmentation (clustering), m/z co-localization, normalization to total ion current (TIC), and lipid annotation against LipidMaps.

Instrumentation used

• MassTech AP-MALDI UHR ion source (MassTech Inc.) enabling rapid swapping between electrospray and MALDI configurations (reported <2 minutes).
• Thermo Scientific Orbitrap Exploris 480 mass spectrometer equipped with EASY-IC for internal lock-mass correction.
• Cryo-Ultramicrotome Leica EM FC6 for 6 μm sectioning.
• SunCollect MALDI-Sprayer for automated matrix deposition.
• Software: MassTech imzML converter and LipostarMSI (v.2.1.0b1) for imaging and lipid annotation.

Main results and discussion

• Spatial segmentation: clustering-based segmentation recovered four main tissue regions that corresponded to RHE histology—stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG) and their interfaces—plus the stratum corneum (SC) and the polycarbonate membrane support.
• Lipid coverage: seven major lipid categories were detected across sections, including glycerolipids, sterols, fatty acyls, glycerophospholipids and sphingolipids. The HRAM Orbitrap allowed confident elemental compositions and annotations (mass accuracy often <1 ppm).
• Biologically relevant localization: 25-hydroxy-cholesterol 3-sulfate (C27H46O5S; m/z 481.2993 [M−H]−) was annotated at 0.6 ppm accuracy. In non-irradiated RHE the ion localized weakly at the SS–SG interface; after UV irradiation the signal increased and localized principally in SB and SS, consistent with UV-induced lipidomic modulation associated with inflammation and cell stress. When RHE was treated with a sun-filter formulation prior to SSR, the localization and intensity of this marker resembled non-irradiated tissue, suggesting protective effect of the formulation.
• Technical performance: AP-MALDI UHR sensitivity and spatial resolution approached vacuum MALDI levels while eliminating pump-down time and facilitating hydrated sample handling. The quick interchangeability with LC-MS workflows on the same instrument enhances experimental throughput and multimodal data integration.

Benefits and practical applications

• Layer-resolved lipid mapping in small and delicate samples such as in vitro skin models enables investigation of cell-layer–specific lipid pathways relevant to barrier function, inflammation, and response to topical formulations.
• High mass accuracy and resolution reduce false annotations and support discovery of low-abundance or isobaric lipid species.
• Rapid switching between electrospray (LC-MS/MS) and AP-MALDI MSI on the same platform streamlines workflows that combine bulk identification/quantitation and spatial localization.
• Applicable to dermatological research, formulation screening (sun-filters, anti-inflammatory actives), biomarker discovery, and QA/QC activities in personal-care product development.

Future trends and potential applications

• Higher-throughput AP-MALDI workflows with faster lasers and automated sample handling will increase dataset sizes and statistical power for spatial omics studies.
• Integration with orthogonal spatial modalities (e.g., spatial transcriptomics, immunohistochemistry) to link lipid distributions with cell-type identity and functional readouts.
• Improved on-tissue MS/MS and ion mobility coupling to resolve isomeric lipids and strengthen structural assignments directly from tissue.
• Application expansion to clinical and translational studies using patient-derived skin models, tape strips, or biopsy sections to map disease-associated lipid signatures at cellular resolution.

Conclusion

The AP-MALDI UHR ion source coupled to an Orbitrap Exploris mass analyzer provides a versatile, high-performance platform for high-spatial-resolution lipidomics in reconstructed human epidermis. The approach yields confident lipid annotations and layer-specific localization of biologically meaningful compounds (e.g., 25-hydroxy-cholesterol 3-sulfate) and can discriminate UV-induced lipid modulation that is mitigated by sun-filter formulations. Rapid interchangeability with LC-MS workflows and the combination of high spatial and mass resolution make this strategy attractive for dermatology research and personal-care product development.

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