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

Mol Cell Proteomics. 2019 Apr;18(4):744-759. doi: 10.1074/mcp.RA118.000958. Epub 2019 Jan 30.


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.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation / drug effects
  • Cell Line
  • Cell Proliferation / drug effects
  • Humans
  • Interferon-gamma / metabolism
  • Mass Spectrometry / methods*
  • Mesenchymal Stem Cells / cytology*
  • Mesenchymal Stem Cells / drug effects
  • Mesenchymal Stem Cells / metabolism
  • Oxygen / pharmacology
  • Proteasome Endopeptidase Complex / metabolism*
  • Reproducibility of Results


  • Interferon-gamma
  • Proteasome Endopeptidase Complex
  • Oxygen