Journal: bioRxiv
Article Title: LipidQMap - An Open-Source Tool for Quantitative Mass Spectrometry Imaging of Lipids
doi: 10.1101/2025.10.15.682422
Figure Lengend Snippet: Sections in panel A. were imaged at 50 × 50 µm, and in panel C. (from the left side): at 50 × 50 µm (Leuven), 17 × 17 µm (Münster), 50 × 50 µm (Dresden), and 20 × 20 µm (Münster). The impact of standard normalization on raw MSI images was evaluated using a glioblastoma mouse brain model via four different mass spectrometry platforms: timsTOF fleX MALDI-2 (Leuven, Belgium), timsTOF fleX MALDI-2 (Münster, Germany), MALDI-Q Exactive TM Orbitrap (Münster, Germany), and AP-SMALDI-Q Exactive TM Orbitrap (Dresden, Germany). All datasets were processed using LipidQMap. A . Comparison of raw MSI images of three lipid species - PC 32:0 [M+H] + , SM 40:1;O2 [M+K] + , and Cer 42:2;O2 [M-H 2 O+H] + - before and after normalization using standards PC [D9] 32:0 [M+H] + , LPC [D9] 16:0 [M+K] + , and Cer 30:1;O2 [M-H 2 O+H] + , respectively. B. Quantitative bulk HILIC-MS/MS lipidomics results from LCM-extracted ROIs as indicated in the brightfield brain scan: blue – brain cortex, orange – tumor rim, red – tumor (glioblastoma), green – corpus callosum. C . Cross-platform comparison of PC 32:0 [M+H] + images in raw ( I. ), isobaric-corrected ( II. ), and standard normalized formats ( III. ). Normalization was performed using PC [D9] 32:0 [M+H] + . The qMSI image of PC 32:0 [M+H] + acquired via MALDI-Q Exactive TM Orbitrap (Münster, Germany), contained empty pixel lines, which were corrected using LipidQMap based on the median lipid concentrations of surrounding pixels.
Article Snippet: MSI SPLASH TM (#330841), PC 15:0/18:1 [D7] (#791637), PC [D5] 17:0/18:1 (#855681), PC [D5] 17:0/16:1 (#855682), PC [D9] 16:0/16:0 (#860352), PE 15:0/18:1 [D7] (#791638), LPC 18:1 [D7] (#791643), SM d18:1;O 2 /18:1 [D9] (#791649), L-CAR [D9] 16:0 (#870323), UltimateSPLASH TM ONE internal standard mix (#330820), SphingoSPLASH TM I internal standard mix (#330734), GM1 18:1;O 2 /18:0 [D7] (#860120), GM3 18:1;O 2 /18:0 [D5] (#860073), GD1b 18:1;O 2 /18:0 [D7] (#860125) were purchased from Avanti Polar Lipids (Alabama, USA), LPC [D9] 16:0 (#L-1516D), LPC [D9] 18:0 (#L-1518D) from Echelon Biosciences (Utah, USA).
Techniques: Mass Spectrometry, Comparison, Hydrophilic Interaction Liquid Chromatography, Tandem Mass Spectroscopy
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![Sections in panels A.-C. were imaged at 30 × 30 µm and H.-L. at 50 × 50 µm. A. Raw MSI image illustrating the isobaric overlap of PC 36:1 [M+Na] + and PC 38:4 [M+H] + and corresponding MS spectrum of monoisotopic signals at 35 000 (red line) and 600 000 (black line) resolution (simulation). B. A raw MSI image showing the overlap of PE O-36:4 [M+Na] + and PE O-38:7 (PE P-38:6) [M+H] + with a comparison of separation capabilities between the same two mass spectrometers (simulation). C. MSI images of PC 36:1 [M+Na] + and PC 38:4 [M+H] + after Na/H adduct isobaric correction of raw images. The top row shows images purified from isobars, the middle row after standard normalization (qMSI image of [M+H] + and [M+Na] + adducts), and the bottom row includes an additional qMSI image of other detected adducts for comparison, normalized to PC [D5] 35:1. D. MSI images of PE O-36:4 [M+Na] + and PE O-38:7 [M+H] + after Na/H adduct isobaric correction of raw images. The top row shows images before standard normalization, the middle row after standard normalization, and the bottom row includes both protonated and sodiated adducts for comparison. Normalization was performed using <t>PE</t> <t>33:1[D7].</t> Isobaric separation was achieved using LipidQMap software, based on the sodium-to-proton adduct ratio of the lipid standard sprayed onto the surface. E . Hematoxylin-stained brain section used for laser capture microdissection. A total area of 5 mm was extracted for each region of interest (ROI) (pooled from multiple sections) for quantitative bulk lipidomics. F, G. Bulk lipidomics results (HILIC-MS/MS) for PC 36:1, PC 38:4, PE O-36:4, and PE P-38:6 obtained from the ROI. The standard deviation was obtained from two technical replicates. H-J. Raw MSI images showing overlapping signals from two lipid adducts: PC 36:1 [M+Na] + and PC 38:4 [M+H] + , acquired on timsTOF fleX MALDI-2, AP-SMALDI Q Exactive TM Orbitrap, DESI-MRT Select Series. K. MSI images after Na/H adduct isobaric correction of raw images by applying the sodium-to-proton adduct ratio of the standard sprayed onto the brain section surface. L. qMSI images after correction of the isobaric overlap between PC 36:1 and PC 38:4 and subsequent standard normalization (PC <t>[D9]</t> 32:0 for both MALDI platforms, and PC [D5] 35:1 for the DESI-qMSI).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_22/10__1101_slash_2025__10__15__682422/10__1101_slash_2025__10__15__682422___F2.large.jpg)
![Sections in panel A. were imaged at 50 × 50 µm, and in panel C. (from the left side): at 50 × 50 µm (Leuven), 17 × 17 µm (Münster), 50 × 50 µm (Dresden), and 20 × 20 µm (Münster). The impact of standard normalization on raw MSI images was evaluated using a glioblastoma mouse brain model via four different mass spectrometry platforms: timsTOF fleX MALDI-2 (Leuven, Belgium), timsTOF fleX MALDI-2 (Münster, Germany), MALDI-Q Exactive TM Orbitrap (Münster, Germany), and AP-SMALDI-Q Exactive TM Orbitrap (Dresden, Germany). All datasets were processed using LipidQMap. A . Comparison of raw MSI images of three lipid species - PC 32:0 [M+H] + , SM 40:1;O2 [M+K] + , and Cer 42:2;O2 [M-H 2 O+H] + - before and after normalization using standards PC [D9] 32:0 [M+H] + , LPC [D9] 16:0 [M+K] + , and Cer 30:1;O2 [M-H 2 O+H] + , respectively. B. Quantitative bulk HILIC-MS/MS lipidomics results from LCM-extracted ROIs as indicated in the brightfield brain scan: blue – brain cortex, orange – tumor rim, red – tumor (glioblastoma), green – corpus callosum. C . Cross-platform comparison of PC 32:0 [M+H] + images in raw ( I. ), isobaric-corrected ( II. ), and standard normalized formats ( III. ). Normalization was performed using PC [D9] 32:0 [M+H] + . The qMSI image of PC 32:0 [M+H] + acquired via MALDI-Q Exactive TM Orbitrap (Münster, Germany), contained empty pixel lines, which were corrected using LipidQMap based on the median lipid concentrations of surrounding pixels.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_22/10__1101_slash_2025__10__15__682422/10__1101_slash_2025__10__15__682422___F5.large.jpg)