Lipidomics reveals dramatic lipid compositional changes in the maturing postnatal lung

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6–8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.


Supplementary Figure Legends
intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). Lines with more than one lipid identification indicate that the listed lipid names were co-eluting. If more than one chromatographic peak was identified with the correct mass, fragmentation pattern, and isotopic profile then the two features (likely isomers) were annotated "lipid_A" and "lipid_B," according to retention time. The underlying numerical data 3 used to generate Figure S3 along with statistical significance values is available in Tables S1 -Tab9. Figure S4. All identified ganglioside (GM3) species shown in heatmap format. Data in heatmap is z-scored and sorted by component 1 of the principal component analysis. Developmental age is shown on the upper x-axis. Each row represents the normalized intensities of a unique chromatographic feature. The features are color coded by row with red indicating high intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). If more than one chromatographic peak was identified with the correct mass, fragmentation pattern, and isotopic profile then the two features (likely isomers) were annotated "lipid_A" and "lipid_B," according to retention time. The underlying numerical data used to generate Figure S4 along with statistical significance values is available in Tables S1 -Tab10.  shown on the upper x-axis. Each row represents the normalized intensities of a unique chromatographic feature. The features are color coded by row with red indicating high intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). Lines with more than one lipid identification indicate that the listed lipid names were co-eluting. If more than one chromatographic peak was identified with the correct mass, fragmentation pattern, and isotopic profile then the two features (likely isomers) were annotated "lipid_A" and "lipid_B," according to retention time. The underlying numerical data used to generate Figure S6 along with statistical significance values is available in Tables S1 -Tab11. Figure S7. Triacylglycerides that are statistically more abundant in the PND7 and PND14 murine lungs had a greater number of double bonds and longer fatty acid chains than those that were most abundant in the adult murine lungs. Data shown are all lipids that were identified as being statistically different (p value < 0.05) between PND7 and adult mice. Developmental age is shown on upper x-axis. Each row represents a unique normalized chromatographic feature. The lipid abundance is color coded with blue indicating low intensity and red indicating high intensity (see color key). Data were sorted based on fold change. Lines with more than one lipid identification indicate that the listed lipid names were co-eluting. Lipids were deleted from the table if the compound was below the limit of detection for any of the replicates. Compounds with more total carbons in the fatty acids are darker green, whereas those with less are lighter.
Compounds with more double bonds in the fatty acids are darker purple, whereas those with less are lighter (see color key). The underlying numerical data used to generate Figure S7 along with statistical significance values is available in Tables S1 -Tab12. 5 Figure S8. The abundances of all identified PE plasmalogen species shown in heatmap form.
Data in heatmap is z-scored and sorted by component 1 of the principal component analysis.
Developmental age is shown on the upper x-axis. Each row represents the normalized intensities of a unique chromatographic feature. The features are color coded by row with red indicating high intensity, blue indicating low intensity, and black indicating below limit of detection (LOD, see color key). Lines with more than one lipid identification indicate that the listed lipid names were co-eluting. If more than one chromatographic peak was identified with the correct mass, fragmentation pattern, and isotopic profile then the two features (likely isomers) were annotated "lipid_A" and "lipid_B," according to retention time. The majority of PE(P-species are more abundant in the adult samples than in the samples undergoing alveolarization. The underlying numerical data used to generate Figure S8 along with statistical significance values is available in Tables S1 -Tab13. Figure S9. The abundances of all identified lysoPC species shown in heatmap form with the same specifications as described in Figure 2. Frequently with lyso species, two peaks are observed chromatographically, commonly thought to be SN-1 and SN-2 isomers of the lyso. If two peaks were able to be identified in this dataset, the peak eluting first was annotated "lipid_A" and the second peak was annotated "lipid_B" as shown above. The majority of LPC species are more abundant in the adult mice than in the mice undergoing alveolarization. The underlying numerical data used to generate Figure S9 along with statistical significance values is available in Tables S1 -Tab14. Figure S10. Beta-hydroxybutyrate is more abundant in PND7 and PND14 murine lungs than adult lungs. Data shown are log 2 normalized abundances of beta-hydroxybutyrate. Error bars show standard error of three replicates. Asterisks denote statistical significance as shown in key. Figure S11. Metabolic pathway of arachidonic acid metabolism (mmu:00590). The entire metabolic pathway is shown to be increased in adults as compared to PND7 and PND14 mice.
Proteins are indicated by rounded rectangles with protein names within, lipids and lipid metabolites are indicated by circles. Red font protein names indicates higher relative abundances were observed in adult samples, green font protein names indicates higher relative abundances were observed in PND7 samples, gray font protein names indicates that the species was not changing in a statistically significant manner. Bar graphs are provided for each protein with significance of differential expression across time indicated by stars. (p<0.001 -***; p<0.01 -**; p<0.05 -*). The entire arachidonic acid metabolic pathway, starting with cleavage from intact phospholipids, is enriched in adults as compared to the younger samples.