Direct Observation of Oligomeric Species formed in the Early Stages of Amyloid Fibril Formation using Electrospray Ionisation Mass Spectrometry - Analytical Methods & Tools for Lyophilization

Direct Observation of Oligomeric Species formed in the Early Stages of Amyloid Fibril Formation using Electrospray Ionisation Mass Spectrometry
Received 20 June 2006; revised 24 August 2006; accepted 28 August 2006. Edited by C. R. Matthews. Available online 1 September 2006.
Andrew M. Smith?, a, Thomas R. Jahn?, a, Alison E. Ashcrofta and Sheena E. Radford, a
Journal of Molecular Biology
Volume 364, Issue 1 , 17 November 2006
ScienceDirect
Copyright ? 2006 Elsevier Ltd All rights reserved.
aAstbury Centre for Structural Molecular Biology, Garstang and Astbury Buildings, University of Leeds, Leeds LS2 9JT, UK
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 (?2m) 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 = 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.
Keywords: amyloid fibril formation; electrospray ionisation mass spectrometry; beta-2-microglobulin; oligomers; analytical ultracentrifugation
Abbreviations: ESI-MS, electrospray ionisation mass spectrometry; ?2m, ?2-microglobulin; AUC, analytical ultracentrifugation; Thio-T, thioflavin-T; AFM, atomic force microscopy
Corresponding author.
? A.M.S. and T.R.J. contributed equally to this work.
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