An optimized approach and inflation media for obtaining complimentary mass spectrometry-based omics data from human lung tissue

Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. To obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated agarose inflation and carboxymethyl cellulose embedding media and determined effective tissue preparation protocols for performing bulk and spatial mass spectrometry-based omics measurements. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. We also compared our mass spectrometry results to lung tissue samples preserved using two other inflation/embedding media, but we identified several pitfalls with the sample preparation steps using this preservation method. Overall, we demonstrated the versatility of the inflation method, and we can start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.

Automated summary

Human disease states are complex and understanding the characteristics of diseases at different levels, cell types, and microanatomical tissue compartments is crucial for a comprehensive understanding of diseases. To accurately observe the molecular landscape in human lungs, it is necessary to use special methods to maintain tissue integrity and molecular stability. By optimizing mass spectrometry imaging methods, unique lipid distributions in different airway regions of the lungs can be observed, and high-resolution proteomic analysis reveals the specific localization of certain proteins in these airway regions. The inflation method shows better results compared to other preservation methods. Through various experiments, we can begin to uncover the spatial connections between the metabolome, lipidome, and proteome in human lungs and across different disease states.