Menneteau, T;
Fabre, B;
Garrigues, L;
Stella, A;
Zivkovic, D;
Roux-Dalvai, F;
Mouton-Barbosa, E;
... Bousquet, M-P; + view all
(2019)
Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells.
Molecular & Cellular Proteomics
10.1074/mcp.RA118.000958.
(In press).
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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.
| Type: | Article |
|---|---|
| Title: | Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1074/mcp.RA118.000958 |
| Publisher version: | https://doi.org/10.1074/mcp.RA118.000958 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| 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 |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10068007 |
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