Proteome turnover analysis in human lung scaffold cultures with stable isotope labelling

Interactions between cells and extracellular matrix (ECM) are crucial for successful reconstruction of tissues from biological scaffolds. It has so far not been possible to distinguish between scaffold and cell derived proteins in mass spectrometry analyses of tissues reconstructed from biological scaffolds. We aimed to develop a method for in-depth proteome analysis of reconstructed lung tissue. Decellularized human lung slices were used as scaffolds and repopulated with allogenic lung fibroblasts in cell media with stable isotope labelled amino acids. Repopulated scaffolds were analysed with tandem mass spectrometry with scaffold proteins and labelled cell derived proteins being quantified simultaneously. With this technique we were able to follow, not only the production of new proteins, but also degradation of scaffold proteins. Fibroblast culture in lung scaffolds led to accumulation of more proteoglycans and an extracellular milieu with greater resemblance to native lung when compared to standard monolayer cultures. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. Stable isotope labelling of synthesised proteins enabled us to quantify low levels of protein synthesis in the tissue cultures, which would otherwise be masked by protein already present in the lung scaffold. The technique presented here expands the usefulness of mass spectrometry-based proteomics in tissue engineering with biological scaffolds, and should be a valuable new tool in understanding cell-ECM interactions.