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Mol Cell Proteomics. 2019 Jan 30. pii: mcp.RA118.000958. doi: 10.1074/mcp.RA118.000958. [Epub ahead of print]

Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells.

Author information

1
IPBS - CNRS - University of Toulouse, France.
2
CNRS/UPS.
3
Centre de Recherche du CHU de Quebec.
4
Institut de Pharmacologie et Biologie Structural (IPBS), CNRS.
5
STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, France.
6
IPBS.
7
IPBS, CNRS UMR 5089, France.
8
IPBS - CNRS - University of Toulouse, France marie-pierre.bousquet@ipbs.fr.

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.

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

PMID:
30700495
DOI:
10.1074/mcp.RA118.000958
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