Stochastic assembly of biomacromolecular complexes: impact and implications on charge interpretation in native mass spectrometry.

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to ∼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.

An in vivo platform to select and evolve aggregation-resistant proteins.

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

An in vivo platform for identifying inhibitors of protein aggregation.

Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a ß-lactamase tripartite fusion construct, that is capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid ß) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split ß-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of islet amyloid polypeptide aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation.

ESI-IMS-MS: A method for rapid analysis of protein aggregation and its inhibition by small molecules.

Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is a powerful method for the study of conformational changes in protein complexes, including oligomeric species populated during protein self-aggregation into amyloid fibrils. Information on the mass, stability, cross-sectional area and ligand binding capability of each transiently populated intermediate, present in the heterogeneous mixture of assembling species, can be determined individually in a single experiment in real-time. Determining the structural characterisation of oligomeric species and alterations in self-assembly pathways observed in the presence of small molecule inhibitors is of great importance, given the urgent demand for effective therapeutics. Recent studies have demonstrated the capability of ESI-IMS-MS to identify small molecule modulators of amyloid assembly and to determine the mechanism by which they interact (positive, negative, non-specific binding, or colloidal) in a high-throughput format. Here, we demonstrate these advances using self-assembly of Aß40 as an example, and reveal two new inhibitors of Aß40 fibrillation.

Insights into the consequences of co-polymerisation in the early stages of IAPP and Aß peptide assembly from mass spectrometry.

The precise molecular mechanisms by which different peptides and proteins assemble into highly ordered amyloid deposits remain elusive. The fibrillation of human amylin (also known as islet amyloid polypeptide, hIAPP) and the amyloid-beta peptide (Aß-40) are thought to be pathogenic factors in Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Amyloid diseases may involve co-aggregation of different protein species, in addition to the self-assembly of single precursor sequences. Here we investigate the formation of heterogeneous pre-fibrillar, oligomeric species produced by the co-incubation of hIAPP and Aß-40 using electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based methods. Conformational properties and gas-phase stabilities of amyloid oligomers formed from hIAPP or Aß40 alone, and from a 1 : 1 mixture of hIAPP and Aß40 monomers, were determined and compared. We show that co-assembly of the two sequences results in hetero-oligomers with distinct properties and aggregation kinetics properties compared with the homo-oligomers present in solution. The observations may be of key significance to unravelling the mechanisms of amyloid formation in vivo and elucidating how different sequences and/or assembly conditions can result in different fibril structures and/or pathogenic outcomes.

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry.

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry-mass spectrometry (IMS-MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS-MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.