title: Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry.
abstract: Numerous debilitating human disorders result from protein misfolding and amyloid formation. Despite the grave nature of these maladies, our understanding of the structural mechanism of fibril assembly is limited. Of paramount importance is the need to identify and characterize oligomeric species formed early during fibril assembly, so that the nature of the initiating assembly mechanism can be revealed and species that may be toxic to cells identified. However, the transient nature of early oligomeric species, combined with their heterogeneity and instability, has precluded detailed analysis to date. Here, we have used electrospray ionisation mass spectrometry (ESI-MS), complemented by analytical ultracentrifugation (AUC) and measurements of thioflavin-T fluorescence, to monitor the early stages of assembly of amyloid-like fibrils formed from human beta-2-microglobulin (beta2m) in vitro. We show that worm-like fibrils that form with nucleation-independent kinetics assemble by a mechanism consistent with monomer addition, with species ranging from monomer to > or = 13-mer being identified directly and uniquely as transient assembly intermediates. By contrast, only monomers, dimers, trimers and tetramers are observed during nucleated growth, which leads to the formation of long straight fibrils. The results highlight the unique power of non-covalent ESI-MS to identify protein assembly intermediates in complex heterogeneous systems and demonstrate its great potential to identify and characterise individual species formed early during amyloid assembly.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Garstang and Astbury Buildings, University of Leeds, Leeds, UK.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Smith
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Jahn
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000544:Alzheimer Disease; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D020836:Protein Structure, Quaternary; D021241:Spectrometry, Mass, Electrospray Ionization; D014461:Ultracentrifugation
chemical_list: D000682:Amyloid