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, Emmanuelle Mouton-Barbosa, Mathilde Beau, Marie-Laure Renoud, François Amalric, et al. To cite this version: Thomas Menneteau, Bertrand Fabre, Luc Garrigues, Alexandre Stella, Dusan Zivkovic, et al.. Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells. Molecular and Cellular Proteomics, American Society for Biochemistry and Molecular Biology, 2019, 18 (4), pp.744-759. 10.1074/mcp.RA118.000958. hal-02167451 HAL Id: hal-02167451 https://hal.archives-ouvertes.fr/hal-02167451 Submitted on 27 Jun 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Research Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells Authors Thomas Menneteau, Bertrand Fabre, Luc Garrigues, Alexandre Stella, Dusan Zivkovic, Florence Roux-Dalvai, Emmanuelle Mouton-Barbosa, Mathilde Beau, Marie-Laure Renoud, Franc¸ois Amalric, Luc Sense´be´ , Anne Gonzalez-de-Peredo, Isabelle Ader, Odile Burlet-Schiltz, and Marie-Pierre Bousquet Correspondence Graphical Abstract Downloaded from [email protected]; [email protected] In Brief 20S proteasomes are very heter- http://www.mcponline.org/ ogeneous protein complexes in- volved in many cellular pro- cesses. In the present study, we combined an MRM-based assay with the production and purifica- tion of entire SILAC labelled pro- by guest on April 15, 2019 teasome to monitor absolute quantities of the different 20S proteasome subtypes in various human cells and tissues. This method applied to adipocyte- derived stem cells (ADSCs) ampli- fied under various conditions highlights an increased expres- sion of immunoproteasome when this type of cell is primed with ␥ IFN or amplified in a 20% O2 environment. Highlights • Design of an MRM assay to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes. • Use of purified isotopically labelled 20S proteasome as internal standard for accurate quantification. • Variation in the expression of immunoproteasome in adipocyte-derived stem cells (ADSCs) grown under different O2 levels might be causal for change in cells differentiation capacity. • The status of 20S proteasome during ADSCs expansion might constitute an additional relevant quality control parameter to contribute to predict, among other quality markers, their therapeutic capacity. Menneteau et al., 2019, Molecular & Cellular Proteomics 18, 744–759 April 2019 © 2019 Menneteau et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. https://doi.org/10.1074/mcp.RA118.000958 los Research Author’s Choice Mass Spectrometry-based Absolute Quantification of 20S Proteasome Status for Controlled Ex-vivo Expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells*□S Thomas Menneteau‡§ʈ, Bertrand Fabre‡ʈ, Luc Garrigues‡, Alexandre Stella‡, Dusan Zivkovic‡, Florence Roux-Dalvai‡, Emmanuelle Mouton-Barbosa‡, Mathilde Beau‡, Marie-Laure Renoud§, Franc¸ois Amalric‡, Luc Sense´be´§, Anne Gonzalez-de-Peredo‡, Isabelle Ader§, Odile Burlet-Schiltz‡¶, and Marie-Pierre Bousquet‡** Downloaded from The proteasome controls a multitude of cellular pro- Some hematopoietic malignancies can be successfully cesses through protein degradation and has been identi- treated by inhibition of the catalytic core 20S proteasome fied as a therapeutic target in oncology. However, our complex, and more recent findings indicate that the protea- understanding of its function and the development of spe- some is a promising target for the treatment of other cancer cific modulators are hampered by the lack of a straight- types or other pathologies including inflammatory diseases http://www.mcponline.org/ forward method to determine the overall proteasome (1–3). status in biological samples. Here, we present a method Although the cylindrical ␣7␤7␤7␣7 barrel-like structure to determine the absolute quantity and stoichiometry of of the 20S catalytic core proteasome has been preserved ubiquitous and tissue-specific human 20S proteasome sub- throughout evolution, the oligomeric protease has evolved, types based on a robust, absolute SILAC-based multiplexed resulting in a higher heterogeneity of subunit compositions in LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sen- mammals. As schematically represented in Fig. 1A, there exist sitivity. The method was initially optimized and validated by at least six distinct forms of 20S proteasomes in human cells by guest on April 15, 2019 1 comparison with a reference ELISA assay and by analyzing and tissues. The standard 20S proteasome (sP20S) is com- the dynamics of catalytic subunits in HeLa cells following posed of constitutive (␣1–␣7 and ␤3, ␤4, ␤6, and ␤7) and IFN␥-treatment and in range of human tissues. It was then catalytic subunits (␤1, ␤2, and ␤5). It is the most abundant ␥ successfully applied to reveal IFN - and O2-dependent vari- 20S subcomplex in most cell types, but significant amounts of ations of proteasome status during primary culture of Adi- other 20S forms have been observed in some human tissues pose-derived-mesenchymal Stromal/Stem Cells (ADSCs). and cells in their basal state or are induced in specific envi- The results show the critical importance of controlling the ronmental conditions (4). For instance, in response to pro- culture conditions during cell expansion for future thera- inflammatory cytokines or in immune cells, the three catalytic peutic use in humans. We hypothesize that a shift from the subunits of the sP20S can be replaced in a highly regulated standard proteasome to the immunoproteasome could way by their immuno counterparts to form the immunopro- serve as a predictor of immunosuppressive and differenti- teasome (iP20S), which has nonidentical cleavage specifici- ation capacities of ADSCs and, consequently, that quality ties (5). Two intermediate proteasomes harboring a mixed control should include proteasomal quantification in addi- assortment of standard and immunocatalytic subunits (␤1i tion to examining other essential cell parameters. The ␤ ␤ method presented also provides a new powerful tool to P20S, 1i 5i P20S) have also been observed in various hu- conduct more individualized protocols in cancer or in- man tissues and cells, and their existence is consistent with flammatory diseases where selective inhibition of the the rules of cooperative assembly of inducible catalytic sub- immunoproteasome has been shown to reduce side units (6). Some other 20S subtypes are much more tissue- effects. Molecular & Cellular Proteomics 18: 744–759, specific, such as the thymoproteasome (containing ␤1i, ␤2i 2019. DOI: 10.1074/mcp.RA118.000958. and ␤5t catalytic subunits) and the spermatoproteasome From the ‡Institut de Pharmacologie et de Biologie Structurale (IPBS), Universite´ de Toulouse, CNRS UMR 5089, UPS, Toulouse, France; §STROMALab, Universite´ de Toulouse, INSERM U1031, EFS, INP-ENVT, UPS, Toulouse, France Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 13, 2018, and in revised form, January 21, 2019 Published, MCP Papers in Press, January 30, 2019, DOI 10.1074/mcp.RA118.000958 744 Molecular & Cellular Proteomics 18.4 © 2019 Menneteau et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. LC-SRM Quantification of 20S Proteasome for ADSCs Expansion (containing a specific isoform of ␣4 called ␣4s) which are SRM has recently been successfully applied in protein assays found in the thymus and male germ cells, respectively. The (16, 17). In these studies, the proteins themselves were not environment- or tissue-specific subunit composition of hu- directly detected and quantified, but were analyzed at peptide man 20S proteasome has been shown to fulfill specialized level, after enzymatic proteolysis. This is a critical step be- functions that the standard proteasome can only exert sub- cause, in most cases, the absolute quantification relies on the optimally (4). For example, immune cells contain a significant addition of an isotopically-labeled peptide standard (called proportion of immunosubunit-containing 20S proteasome AQUA peptide) (18) late in the experimental workflow, which subtypes which display specific proteolytic preferences might introduce a high variability because of differences in thanks to which they produce antigens for presentation to sample preparation. Thus, for accurate and robust absolute CD8 T cells (6). In the thymus, the unique cleavage preference quantification of proteins, and to determine protein stoichi- of ␤5t explains the essential role