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Molecular & Cellular Proteomics

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

View ORCID ProfileThomas Menneteau, Bertrand Fabre, Luc Garrigues, Alexandre Stella, Dusan Zivkovic, Florence Roux-Dalvai, Emanuelle Mouton-Barbosa, Mathilde Beau, Marie-Laure Renoud, François Amalric, Luc Sensebe, Anne Gonzalez-de-Peredo, Isabelle Ader, Odile Burlet-Schiltz and View ORCID ProfileMarie-Pierre Bousquet  Correspondence email
Molecular & Cellular Proteomics January 30, 2019, mcp.RA118.000958; https://doi.org/10.1074/mcp.RA118.000958
Thomas Menneteau
IPBS - CNRS - University of Toulouse, France
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Bertrand Fabre
IPBS - CNRS - University of Toulouse, France
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Luc Garrigues
IPBS - CNRS - University of Toulouse, France
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Alexandre Stella
CNRS/UPS
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Dusan Zivkovic
IPBS - CNRS - University of Toulouse, France
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Florence Roux-Dalvai
Centre de Recherche du CHU de Quebec
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Emanuelle Mouton-Barbosa
Institut de Pharmacologie et Biologie Structural (IPBS), CNRS
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Mathilde Beau
IPBS - CNRS - University of Toulouse, France
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Marie-Laure Renoud
STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, France
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François Amalric
IPBS - CNRS - University of Toulouse, France
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Luc Sensebe
STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, France
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Anne Gonzalez-de-Peredo
IPBS
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Isabelle Ader
STROMALab, Université de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, France
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Odile Burlet-Schiltz
IPBS, CNRS UMR 5089, France
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Marie-Pierre Bousquet
IPBS - CNRS - University of Toulouse, France
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  • For correspondence: marie-pierre.bousquet@ipbs.fr
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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.

  • 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.
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Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells
Thomas Menneteau, Bertrand Fabre, Luc Garrigues, Alexandre Stella, Dusan Zivkovic, Florence Roux-Dalvai, Emanuelle Mouton-Barbosa, Mathilde Beau, Marie-Laure Renoud, François Amalric, Luc Sensebe, Anne Gonzalez-de-Peredo, Isabelle Ader, Odile Burlet-Schiltz, Marie-Pierre Bousquet
Molecular & Cellular Proteomics January 30, 2019, mcp.RA118.000958; DOI: 10.1074/mcp.RA118.000958

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Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells
Thomas Menneteau, Bertrand Fabre, Luc Garrigues, Alexandre Stella, Dusan Zivkovic, Florence Roux-Dalvai, Emanuelle Mouton-Barbosa, Mathilde Beau, Marie-Laure Renoud, François Amalric, Luc Sensebe, Anne Gonzalez-de-Peredo, Isabelle Ader, Odile Burlet-Schiltz, Marie-Pierre Bousquet
Molecular & Cellular Proteomics January 30, 2019, mcp.RA118.000958; DOI: 10.1074/mcp.RA118.000958
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