@article{discovery10068007,
            year = {2019},
         journal = {Molecular \& Cellular Proteomics},
            note = {This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions.},
           title = {Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells},
        keywords = {20S Proteasome, Absolute quantification, Human Adipose-derived Mesenchymal Stromal/Stem Cells, Protein complex analysis, SILAC, Selected reaction monitoring, Stem cells*, Targeted mass spectrometry, stoichiometry},
          author = {Menneteau, T and Fabre, B and Garrigues, L and Stella, A and Zivkovic, D and Roux-Dalvai, F and Mouton-Barbosa, E and Beau, M and Renoud, M-L and Amalric, F and Sensebe, L and Gonzalez-de-Peredo, A and Ader, I and Burlet-Schiltz, O and Bousquet, M-P},
            issn = {1535-9484},
             url = {https://doi.org/10.1074/mcp.RA118.000958},
        abstract = {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 IFN{\ensuremath{\gamma}}-treatment and in range of human tissues. It was then successfully applied to reveal IFN{\ensuremath{\gamma}}- 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.}
}