IPBS-CNRS - SRM_Proteasome_2018

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

  • Organism: Homo sapiens
  • Instrument: QTRAP 5500, QTRAP 6500
  • SpikeIn: No
Abstract
Proteasome controls a plethora of cellular processes through protein degradation and is a therapeutic target in oncology. However, dissection of its function and development of specific modulators are hampered by the lack of a straightforward method to determine the full proteasome status in biological samples. The presented approach describes how the copy number and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes can be obtained in a single, robust, absolute SILAC-based multiplexed LC-SRM mass spectrometry assay with high precision, accuracy, and sensitivity. The developed method was initially optimized and validated by comparison to an existing ELISA reference assay and by analyzing the dynamics of catalytic subunits in response to IFNgamma and in various human tissues. It was then successfully applied to reveal IFNgamma- 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 human. It is proposed that a shift from standard proteasome to immunoproteasome could serve as a predictor of immunosuppressive and differentiation abilities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to other essential cell parameters. The developed method also provides a new powerful tool for conducting more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has proven to reduce side-effects.
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Created on 7/12/18 10:01 AM