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.
- stoichiometry
- Human Adipose-derived Mesenchymal Stromal/Stem Cells
- 20S Proteasome
- Absolute quantification
- Protein complex analysis
- Selected reaction monitoring
- SILAC
- Stem cells*
- Targeted mass spectrometry
Footnotes
Author contributions: T.M., B.F., L.G., A.S., D.Z., F.R.-D., M.B., M.-L.R., A.G.-dP., I.A., and M.-P.B. performed research; T.M., B.F., L.G., E.M.-B., A.G.-dP., I.A., and M.-P.B. analyzed data; F.A., I.A., O.B.-S., and M.-P.B. wrote the paper; L.S. and I.A. contributed new reagents/analytic tools; A.G.-dP., I.A., O.B.-S., and M.-P.B. designed research.
- Received July 13, 2018.
- Revision received January 21, 2019.
- Accepted January 30, 2019.
- Published under license by The American Society for Biochemistry and Molecular Biology, Inc.