Simultaneous monitoring of peptide aggregate distributions, structure, and kinetics using amide hydrogen exchange: Application to Aβ(1-40) fibrillogenesis

Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22904, USA.
Biotechnology and Bioengineering (Impact Factor: 4.16). 08/2008; 100(6):1214-1227. DOI: 10.1002/bit.21846

ABSTRACT Increasing evidence indicates that soluble aggregates of amyloid beta protein (Abeta) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Abeta and other aggregating peptides and proteins. In this study, hydrogen-deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Abeta(1-40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX-MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Abeta aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30-50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Abeta toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX-MS approach to characterizing complex, multi-component oligomer distributions of aggregating peptides and proteins.

Download full-text


Available from: Dhara A Patel, Dec 26, 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Amyloid-β peptide (Aβ) varies in size from 39 to 43 amino acids and arises from sequential β- and γ-secretase processing of the amyloid precursor protein. Whereas the non-pathological role for Aβ is yet to be established, there is no disputing that Aβ is now widely regarded as central to the development of Alzheimer's disease (AD). The so named "amyloid cascade hypothesis" states that disease progression is the result of an increased Aβ burden in affected areas of the brain. To elucidate the Aβ role in AD, many analytical approaches have been proposed as suitable tools to investigate not only the total Aβ load but also many other issues that are considered crucial for AD, such as: (i) the aggregation state in which Aβ is present; (ii) its interaction with other species or metals; (iii) its ability to induce oxidative stress; and (iv) its degradative pathways. This review provides an insight into the use of mass spectrometry (MS) in the field of Aβ investigation aimed to assess its role in AD. In particular, the different MS-based approaches applied in vitro and in vivo that can provide detailed information on the above-mentioned issues are reviewed. Moreover, the advantages offered by the MS methods over all the other techniques are highlighted, together with the recent developments and uses of combined analytical approaches to detect and characterize Aβ.
    Mass Spectrometry Reviews 05/2011; 30(3):347-65. DOI:10.1002/mas.20281
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: beta-Amyloid (Abeta) peptide is believed to play a key role in the mechanism of Alzheimer's disease (AD). Abeta tends to aggregate to form amyloid fibrils. A variety of evidence indicates that Abeta aggregates are toxic in vitro and in vivo. An early "Abeta hypothesis" postulated that AD was the consequence of neuron death induced by insoluble deposits of large Abeta fibrils. Newer findings indicate that small soluble Abeta oligomers are the neurotoxic species, yet their structure is still unknown. Many researchers have tried to probe the differences in molecular structure between Abeta oligomers, protofibrils, and fibrils that give rise to their unique toxicities, but with limited success. In this report, we examine the hypothesis that differences in the toxicity of different aggregated Abeta species are the result of differences in species concentration and diffusivity. Using a simple mathematical analysis based on the assumption of a diffusion-limited reaction, we demonstrate that near 10-fold differences in toxicity between spherical oligomers and fibrils can be explained from size and concentration arguments. While this work does not suggest that Abeta oligomers and fibrils have identical molecular structures, it highlights the possibility that simple physical phenomena may contribute to the biological processes induced by Abeta.
    Biotechnology and Bioengineering 06/2010; 106(2):333-7. DOI:10.1002/bit.22691
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Beta-amyloid peptide (Abeta) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Abeta could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide-membrane interactions of a model beta-sheet peptide, P(11-2) (CH(3)CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH(2)), by fluorescence, infrared spectroscopy, and hydrogen-deuterium exchange. Like Abeta(1-40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P(11-2) also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial beta-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model beta-sheet peptide recapitulates many important features of Abeta peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.
    Biochimica et Biophysica Acta 05/2009; 1788(9):1714-21. DOI:10.1016/j.bbamem.2009.04.010