Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

20S proteasomes are very heterogeneous protein complexes involved in many cellular processes. In the present study, we combined an MRM-based assay with the production and purification of entire SILAC labelled proteasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte-derived stem cells (ADSCs) amplified under various conditions highlights an increased expression of immunoproteasome when this type of cell is primed with IFNγ or amplified in a 20% O2 environment.
Graphical Abstract Highlights Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O2 levels might be causal for change in cells differentiation capacity. The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity.
The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects.