Citation: Dafun, A.S.; Živkovi´ c, D.; Leon-Icaza, S.A.; Möller, S.; Froment, C.; Bonnet, D.; de Jesus, A.A.; Alric, L.; Quaranta-Nicaise, M.; Ferrand, A.; et al. Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS. Cells 2023, 12, 844. https://doi.org/ 10.3390/cells12060844 Academic Editors: Marcus Groettrup and Michael Basler Received: 5 December 2022 Revised: 23 February 2023 Accepted: 27 February 2023 Published: 8 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS Angelique Sanchez Dafun 1 , Dušan Živkovi´ c 1 , Stephen Adonai Leon-Icaza 1 , Sophie Möller 2 , Carine Froment 1 , Delphine Bonnet 3,4 , Adriana Almeida de Jesus 5 , Laurent Alric 4 , Muriel Quaranta-Nicaise 3 , Audrey Ferrand 3 ,Céline Cougoule 1 , Etienne Meunier 1 , Odile Burlet-Schiltz 1 , Frédéric Ebstein 2,† , Raphaela Goldbach-Mansky 5 , Elke Krüger 2 , Marie-Pierre Bousquet 1, * and Julien Marcoux 1, * 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: marie.pierre-bousquet@ipbs.fr (M.-P.B.); julien.marcoux@ipbs.fr (J.M.) † Present address: L’institut du Thorax, Nantes Université, Inserm UMR 1087/CNRS UMR 6291, 44000 Nantes, France. Abstract: The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and β1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifi- cations (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1–2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits. Keywords: immunoproteasome; PRAAS; proteasome; proteoform; top-down proteomics; label-free quantification 1. Introduction During evolution, the constitutive catalytic proteasome subunits (c20S) β1/β2/β5 gave rise to the catalytic immunoproteasome (i20S) subunits β1i/β2i/β5i [1]. Interme- diate forms also exist, in which only one or two constitutive catalytic subunits are re- placed [2]. The incorporation of these alternative immunoproteasome isoforms, which share ~60–70% sequence identity with their standard counterparts, modulates proteasome Cells 2023, 12, 844. https://doi.org/10.3390/cells12060844 https://www.mdpi.com/journal/cells