Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.

The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and ß1-7) 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.

Structural mass spectrometry of membrane proteins.

The analysis of proteins and protein complexes by mass spectrometry (MS) has come a long way since the invention of electrospray ionization (ESI) in the mid 80s. Originally used to characterize small soluble polypeptide chains, MS has progressively evolved over the past 3 decades towards the analysis of samples of ever increasing heterogeneity and complexity, while the instruments have become more and more sensitive and resolutive. The proofs of concepts and first examples of most structural MS methods appeared in the early 90s. However, their application to membrane proteins, key targets in the biopharma industry, is more recent. Nowadays, a wealth of information can be gathered from such MS-based methods, on all aspects of membrane protein structure: sequencing (and more precisely proteoform characterization), but also stoichiometry, non-covalent ligand binding (metals, drug, lipids, carbohydrates), conformations, dynamics and distance restraints for modelling. In this review, we present the concept and some historical and more recent applications on membrane proteins, for the major structural MS methods.