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Publication Detail
Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Article
  • Authors:
    Menneteau T, Fabre B, Garrigues L, Stella A, Zivkovic D, Roux-Dalvai F, Mouton-Barbosa E, Beau M, Renoud M-L, Amalric F, Sensebe L, Gonzalez-de-Peredo A, Ader I, Burlet-Schiltz O, Bousquet M-P
  • Publication date:
    30/01/2019
  • Journal:
    Mol Cell Proteomics
  • Status:
    Published online
  • Country:
    United States
  • PII:
    RA118.000958
  • Language:
    eng
  • 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
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 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.
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