Exposing structural features of protein assemblies using mass spectrometry

Abstract

Most cellular processes depend on a multitude of molecular machines, predominantly comprised of proteins. The diverse set of tasks performed by these machines, known as multi-proteoform complexes (MPCs), is driven by interactions between heterogeneous sets of proteoforms, products of the multi-stage process of protein synthesis and maturation. To understand the biological processes driven by the MPCs, researchers attempt to characterize both the involved proteoforms individually and the entire protein assemblies in-depth with various techniques capable of uncovering structural details. While some structural methods provide a high-resolution and near-atomic structural snapshot, others allow researchers to infer specific structural features in either a high-throughput or a targeted fashion at a lower resolution. Mass spectrometry has emerged in the last decades as a versatile and highly complementary technique with respect to established high-resolution structural methods, e.g. cryo-EM. With mass spectrometry, it is possible to transfer proteins from their natural in-solution environment to the gas-phase, predominantly by electrospray ionization (ESI), and separate them based on their mass-to-charge (m/z) ratio. In a secondary, or tandem, step ions selected based on their m/z ratios can be activated, which results in the disruption of covalent and/or non-covalent bonds, producing characteristic dissociation products. In this thesis, the versatility and power of mass spectrometry applied to intact MPCs are demonstrated by several experimental approaches, sub-divided in two major sections: (I) the structural analysis of MPCs through gas-phase activation, and (II) the in-depth characterization of sophisticated multi-proteoform assemblies with hybrid MS-based approaches.