High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs

Abstract

Mass spectrometry imaging (MSI) provides label-free, non-targeted molecular and spatial information of the biomolecules within tissue. Lipids play important roles in lung biology, e.g. as surfactant, preventing alveolar collapse during normal and forced respiration. Lipidomic characterization of late fetal mouse lungs at day 19 of gestation (E19) has not been performed yet. In this study we employed high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization MSI for the lipidomic analysis of E19 mouse lungs. Molecular species of different lipid classes were imaged in E19 lung sections at high spatial and mass resolution in positive- and negative-ion mode. Lipid species were characterized based on accurate mass and on-tissue tandem mass spectrometry. In addition, a dedicated sample preparation protocol, homogenous deposition of matrices on tissue surfaces and data processing parameters were optimized for the comparison of signal intensities of lipids between different tissue sections of E19 lungs of wild type and Pex11β-knockout mice. Our study provides lipid information of E19 mouse lungs, optimized experimental and data processing strategies for the direct comparison of signal intensities of metabolites (lipids) among the tissue sections from MSI experiments. To best of our knowledge, this is the first MSI and lipidomic study of E19 mouse lungs.

Figure 1

Optimized MSI workflow for characterization and comparative lipidomic analysis of late fetal mouse lung sections at day 19 of gestation (E19) using atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI). (A) Breeding of WT and Pex11β KO E19 animals. (B) Cryosectioning of fresh snap-frozen E19 mouse lung tissues for MSI experiments using a cryomicrotome. (C) Optical phase contrast image of a 12 µm thick section of E19 mouse lung and selection of optimal sections. (D) Tissue section fixed to the sample probe. (E) Homogenous deposition of matrices using an automatic pneumatic ultrafine sprayer (“SMALDIPrep”). (F) MSI data acquisition using an “AP-SMALDI10” ion source coupled to a Q Exactive mass spectrometer. (G) MSI data sets (.raw files). (H) Characterization of E19 (WT) mouse lung lipidome. (I) Optimization of the data analysis workflow for comparative lipidomic analysis. (J) Comparison of relative signal intensities of lipids between WT and Pex11β KO E19 lung tissue sections.

Figure 2

Single-pixel (10 µm) mass spectrum obtained from late fetal E19 mouse lung mass spectrometry imaging (MSI) experiments. (A) Negative-ion single-pixel mass spectrum for mass range m/z 650–850. (B) Positive-ion single-pixel mass spectrum for mass range m/z 700–850. Different classes of lipid species were identified based on high mass accuracy (≤2 ppm), labelled with measured mass and charge carrier.

Figure 3

Characterization of late fetal E19 mouse lung lipidome using high-resolution AP-SMALDI mass spectrometry imaging experiments. (A,E) Microscopic images of E19 mouse lung. (B–D) Negative-ion MS images. (B) [PA(36:2) − H]⁻, (C) [PE(34:1) − H]⁻, (D) [PS(38:4) − H]⁻. (F–H) Positive-ion MS images. (F) [PC(36:5) + K]⁺, (G) [TG(50:1) + K]⁺, (H) [SM(34:1) + K]⁺. Scale bar 500 µm.

Figure 4

Scheme of the optimized data processing framework.

Figure 5

Distribution of matrix cluster signals in wild type (WT) and Pex11β knockout (KO) late fetal E19 mouse lung tissue sections from mass spectrometry imaging (MSI) experiments. (A–C) Evenly distributed 4-Nitroaniline (pNA) matrix cluster images in negative-ion mode. (D–F) Evenly distributed 2,5-Dihydroxybenzoic acid (DHB) matrix cluster images in positive-ion mode. Scale bar 500 µm.

Figure 6

Homogenous distribution of endogenous compounds in wild type (WT) and PEX11β knockout (KO) late fetal E19 mouse lung tissue sections, measured by atmospheric-pressure MALDI MSI experiments. (A-C) Evenly distributed lipid species in negative-ion mode. (A) [PE-Cer(36:1) − H]⁻, (B) [PE(32:0) − H]⁻ and (C) [CerP(34:1) − H]⁻. (D–F) Evenly distributed lipid species in positive-ion mode. (D) [PE(36:3) + K]⁺, (E) [PE(36:4) + Na]⁺ and (F) [PE(34:2) + Na]⁺. Scale bar 500 µm.

Figure 7

Differential abundance of lipid species in wild type (WT) and Pex11β knockout (KO) late fetal E19 mouse lung tissue sections in negative- and positive-ion mode. Downregulation of [PG(34:1) − H]⁻ lipid (m/z 747.51815), upregulation of [SHexCer(t33:1) + K]⁺ lipid (m/z 820.46415) in Pex11β KO E19 mouse lungs in negative- and positive-ion mode. The abundance comparisons between lipids in different cryosections were verified by applying various normalization algorithms: (i) no normalization, (ii) total ion count (TIC) normalization, (iii) normalization to homogenously distributed matrix signals and (iv) normalization to homogenously distributed endogenous signals. Scale bar 500 µm.

Figure 8

Confirmation of differential abundance of lipid species in wild type (WT) and Pex11β knockout (KO) late fetal E19 mouse lung tissue sections for various charge carriers in positive-ion mode. (A–C) Differential abundance of LPC(14:0) in WT and KO E19 mouse lung tissue. (A) [LPC(14:0) + H]⁺, (B) [LPC(14:0) + Na]⁺, (C) [LPC(14:0) + K]⁺. (D–F) Differential abundance of LPC(16:0) in WT and KO E19 mouse lung tissue. (D) [LPC(16:0) + H]⁺, (E) [LPC(16:0) + Na]⁺, (F) [LPC(16:0) + K]⁺. Scale bar 500 µm.

Figure 9

Structural confirmation of lipid annotations by on-tissue tandem mass spectrometry (MS/MS) in negative- and positive-ion mode, using high-energy collisional dissociation (HCD). (A) On-tissue HCD-MS/MS (averaged) spectrum of [PG(34:1) − H]⁻ m/z 747.51966 ± 0.4. (B) On-tissue HCD-MS/MS (averaged) spectrum of [LPC(16:0) + H]⁺ m/z 496.34007 ± 0.4.