MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics

We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid–protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.


Spraying parameters
The internal standard dilution was sprayed with an off-line 2.5 mL syringe (Trajan Scientific, Victoria, Australia) in combination with a syringe pump connected to a HTX TM-Sprayer (HTX Technologies LLC).Spraying parameters for the internal standard were as followed: temperature = 30°C, flow rate = 0.06 mL/min, velocity = 1200 mm/min, track spacing = 3 mm, gas flow = 2 L/min, N2 gas pressure = 10 psi nozzle height = 40 mm, drying time = 30 s, number of passes = 16, in a C-C pattern.The concentration of the internal standard in picomole per squared millimeter (pmol/mm2) was calculated by multiplying the concentration of the internal standard (µg/mL), flow rate (mL/min), time (min), number of passes (layers) divided by the surface area sprayed (mm2).
This number was then divided by dilution factor and the molecular weight (Da), resulting in a value in pmol/mm2.

Hematoxylin and eosin staining
Hematoxylin and eosin (H&E) staining was performed on consecutive sections to the sections used in the MALDI-MSI experiments.The H&E staining procedure was performed as followed: the slides were hydrated in water for 1 minute, stained in hematoxylin for 3 minutes, rinsed with running tap water for 3 minutes, stained in eosin staining for 30 seconds, and again rinsed with running tap water for 3 minutes.The slides were then immersed in 100% ethanol for 1 minute, afterwards xylene washed for 2 minutes, and Entellan-mounted carefully with a coverslip, and left to air-dry at room temperature.All optical images were captured at high resolution using the Leica AperioCS2 scanner (20x objective) via Aperio ImageScope software (version 12.4.3.5008) from Leica Biosystems Imaging (Germany).

Laser Capture Microdissection (LMD)
The coordinate information of the ROIs were exported via LipostarMSI to a Leica LMD 7000 (Leica Microsystems) for laser capture microdissection.The ROIs were dissected using the following laser parameters: power = 54, aperture = 10, speed = 30, specimen balance = 25, line spacing = 5, head current at 100% and pulse frequency at 250 Hz in draw + scan mode for ITO-slides and Intellislides.Laser parameters for PEN-membrane slides were power = 40, aperture = 7, speed = 10, specimen balance = 0, line spacing = 29, head current at 100% and pulse frequency at 2600 Hz in draw + cut mode.Directly after dissection the tissue was collected in the cap of 0.5 mL sterilized PCR-tubes, pre-filled with 20 µL MeOH.The dissected material was transferred to a 2 mL Eppendorf tube and the cap was washed four times with 20 µL MeOH, for a total of 100 µL MeOH.

Supplementary table 4
Significantly altered Metaboanalyst metabolite-gene pathways from the comparison of the tumor region versus the necrotic region.Proteins and lipids used in the joint-pathway analysis were considered up-or downregulated for the specific region (logFC ≥0.58 and logFC ≤-0.58 respectively and with a p-value of ≤0.05).

Supplementary table 5
Significantly altered proteins and lipids used in the comparison of the tumor region versus the necrotic region.Proteins and lipids were considered up-or downregulated for the specific region (logFC ≥0.58 and logFC ≤-0.58 respectively and with a pvalue of ≤0.05).

Name Molecular Formula Average Mass (Da) [ISTD] [Sprayed] [Tissue] (μg/mL) (μg/mm2) pmol/mm2 15:0-18:1 (d7) PC
Number of identified region-specific lipids per lipid class on different slide types.Lipid adducts and isotopes are removed from the data set.Lipids had to be found in at least two samples related to the ROI and slide to be included for lipid identification.List of lipids and their quantification in pmol/mm 2 for comparing Q-MSI and Q-LC-MS between different slide types and tissue regions.Concentrations are a sum of the [M+H] + , [M+Na] + and [M+K 39 ] + adducts.The corresponding internal standard is shown for each lipid class.N.F.= Lipid not found in Lipostar LC-MS identification database.