Angelique Sanchez Dafun

Establishing proteoform footprints of 20S proteasomes from biological and patient samples using top-down mass spectrometry

Angelique Sanchez Dafun1; Dušan Živković1; Stephen Adonai Leon Icaza1; Sophie Möller2; Carine Froment1; Delphine Bonnet3,4; Adriana Almeida De Jesus5; Laurent Alric4; Muriel Quaranta-Nicaise3; Audrey Ferrand3; Céline Cougoule1; Etienne Meunier1; Odile Burlet-Schiltz1; Frédéric Ebstein2,†; Raphaela Goldbach-Mansky5; Elke Krüger2; Marie-Pierre Bousquet1; Julien Marcoux1

1 Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
2 Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, 17475 Greifswald, Germany
3 IRSD, Université de Toulouse, INSERM, INRAE, ENVT, Université de Toulouse III—Paul Sabatier (UPS), 31300 Toulouse, France
4 Internal Medicine Department of Digestive Disease, Rangueil Hospital, Université de Toulouse III—Paul Sabatier (UPS), 31400 Toulouse, France
5 Translational Autoinflammatory Diseases Section, LCIM, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA

* Correspondence: (M.-P.B.); (J.M.) † Present address: L’institut du Thorax, Nantes Université, Inserm UMR 1087/CNRS UMR 6291, 44000 Nantes, France.

Introduction: Proteasome (20S) is the main responsible for protein degradation within the cells, making it a good therapeutic target for some diseases. Its activity is altered by post-translational modifications (PTM), genetic variations, and replacement of its catalytic subunits. Due to its complexity and heterogeneity, these informations are difficult to obtain through immunochemical methods and classical peptide-based approaches. To address this, we developed a miniaturized workflow combining top-down MS (TD-MS) and bottom-up MS (BU-MS) of immunopurified 20S providing their assembly status and proteoform footprint, revealing PTMs, mutation, single-nucleotide-polymorphisms (SNP) and induction of alternative subunits in different samples: organoids, biopsies, B-lymphoblastoid cell lines (BLCL) from Proteasome-Associated-Autoinflammatory-Syndrome (PRAAS) patients. This technique enables better description of 20S, which can later lead to more targeted therapies.

Methods: Proteasome complexes were immunopurified from cell lysates and analyzed by online nano-LC (UltiMate-3000) coupled with an Orbitrap Fusion Tribrid (Thermo). Each sample (1-2 pmol) was injected onto a reverse-phase C4-precolumn (Thermo) and separated on an in-house analytical C4 nanocolumn. MS and MSMS (EThcD) scans were acquired in positive and intact protein modes, at 7,500 and 60,000 resolution, respectively. MS spectra were deconvoluted using RoWinPro and visualized with VisioProtMS. MSMS data was processed in Proteome Discoverer 2.3 (Thermo) using a three-tier search to identify the proteoforms from a custom database. Label-free quantification of 20S subunits was done with deconvoluted results from UniChrom by applying correction using relative ionization yield factors obtained beforehand, from pure constitutive (c20S) and immunoproteasome (i20S).

Preliminary Data: We demonstrated the benefits and current limitations of 20S immunopurification followed by TD-MS to unravel the proteoforms of 20S subunits and to gain information on their PTMs, PRAAS-related mutations, common SNPs and immuno subunits on IFNγ-induced Caco2 cells, lung organoid and intestinal crypts. We identified a novel triphosphorylated and oxidized proteoform of α7 subunit in different biological samples that is independent of 20S subtype and is highly unstable, which likely explains why conventional BU-MS and immunoblotting techniques were unable to detect it so far. With our approach combining TD-MS and BU-MS, we were able to confirm mutations in BLCLs produced from PRAAS patients, which provided insights about the potential impact of these mutations on 20S assembly. In addition, we were able to track the i20S subunits in samples with increasing levels of complexity, including cell lines, organoids, and biopsies. We noticed that 1-3 mg of total protein were sufficient as a starting point to establish these 20S proteoform footprints, suggesting that this strategy can be used with rare biological samples such as disease-related biopsies. We also investigated the potential of TD-MS for intra-acquisition relative quantification of different subunits to establish the proportion of c20S vs. i20S in biological samples. We confirmed that the relative ionization yields (RIY) of the different subunits were quite different (30% to 160% relative to the average of all the non-catalytic subunits), but reproducible. This allowed us to semi-quantify the relative amount of catalytic subunits after induction of the i20S in Caco2 cells, colorectal crypts from patients, and BLCLs produced from PRAAS patients. Even though the standard deviations were not negligible, the obtained values were in good agreement with the expected ratios from label-free BU-MS, showing that the correction of TD-MS abundances with RIY can be used to determine the relative abundance of c20S vs. i20S proteasomes.

Novel Aspect: Characterization of new proteasome proteoforms; Analysis of proteasome from disease-related biopsies; Label-free quantification using relative ionization yields

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