학술논문
Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.
Document Type
Article
Author
Dafun, Angelique Sanchez; Živković, Dušan; Leon-Icaza, Stephen Adonai; Möller, Sophie; Froment, Carine; Bonnet, Delphine; de Jesus, Adriana Almeida; Alric, Laurent; Quaranta-Nicaise, Muriel; Ferrand, Audrey; Cougoule, Céline; Meunier, Etienne; Burlet-Schiltz, Odile; Ebstein, Frédéric; Goldbach-Mansky, Raphaela; Krüger, Elke; Bousquet, Marie-Pierre; Marcoux, Julien
Source
Subject
*PROTEASOMES
*SINGLE nucleotide polymorphisms
*GENETIC variation
*POST-translational modification
*MASS spectrometry
*CELL lines
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Language
ISSN
2073-4409
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 modifications (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. [ABSTRACT FROM AUTHOR]