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 Aβ40 as an example, and reveal two new inhibitors of Aβ40 fibrillation

The Role of Initial Oligomers in Amyloid Fibril Formation by Human Stefin B

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation

Novel insights into protein misfolding diseases revealed by ion mobility-mass spectrometry

Amyloid disorders incorporate a wide range of human diseases arising from the failure of a specific peptide or protein to adopt, or remain in, its native functional conformational state. These pathological conditions, such as Parkinson's disease, Alzheimer's disease and Huntington's disease are highly debilitating, exact enormous costs on both individuals and society, and are predicted to increase in prevalence. Consequently, they form the focus of a topical and rich area of current scientific research. A major goal in attempts to understand and treat protein misfolding diseases is to define the structures and interactions of protein species intermediate between fully folded and aggregated, and extract a description of the aggregation process. This has proven a difficult task due to the inability of traditional structural biology approaches to analyze structurally heterogeneous systems. Continued developments in instrumentation and analytical approaches have seen ion mobility-mass spectrometry (IM-MS) emerge as a complementary approach for protein structure determination, and in some cases, a structural biology tool in its own right. IM-MS is well suited to the study of protein misfolding, and has already yielded significant structural information for selected amyloidogenic systems during the aggregation process. This review describes IM-MS for protein structure investigation, and provides a summary of current research highlighting how this methodology has unequivocally and unprecedentedly provided structural and mechanistic detail pertaining to the oligomerization of a variety of disease related proteins.Danielle M. Williams and Tara L. Pukal