Aβ amyloid fibrils possess a core structure highly resistant to hydrogen exchange

Abstract

We describe here experiments designed to characterize the secondary structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide A␤, using hydrogen-deuterium exchange measurements evaluated by mass spectrometry. The results show that Ϸ50% of the amide protons of the polypeptide backbone of A␤(1-40) resist exchange in aqueous, neutral pH buffer even after more than 1,000 h of incubation at room temperature. We attribute this extensive, strong protection to H-bonding by residues in core regions of ␤-sheet structure within the fibril. The backbone amide hydrogens exchange at variable rates, suggesting different degrees of protection within the fibril. These data suggest that it is unlikely that the entire A␤ sequence is involved in H-bonded secondary structure within the amyloid fibril. Future studies using the methods described here should reveal further details of A␤ fibril structure and assembly. These methods also should be amenable to studies of other amyloid fibrils and protein aggregates.

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

“…The extent of incorporation of 18 O into Aβ in experiments involving H 2 18 O was estimated by treating the experimental isotopic cluster as a linear combination of the cluster obtained with unlabeled water, and a hypothetical cluster for singly 18 O-labeled Aβ. The latter was modeled by shifting each peak in the unlabeled cluster upward by 2 Da (shifting m/z upwards by 2/z).…”

“…To probe this chemistry, the experiment was repeated using 18 O-labeled water. Specifically, 4 µM Aβ monomer in 56/44/0.5 (v/v/v) H 2 O/H 2 18 O/HCOOH was pumped at 5 µl/min in Tube 1 in the triaxial mode, while 100/0.5 (v/v) MeCN/HCOOH was pumped at 5 µl/min in Tube 2, leading to a final water composition that was 44.1% 18 O.…”

“…Specifically, 4 µM Aβ monomer in 56/44/0.5 (v/v/v) H 2 O/H 2 18 O/HCOOH was pumped at 5 µl/min in Tube 1 in the triaxial mode, while 100/0.5 (v/v) MeCN/HCOOH was pumped at 5 µl/min in Tube 2, leading to a final water composition that was 44.1% 18 O. The oxidized peaks in the resulting mass spectrum (averaged over +4, +5, and +6 charge states) contained 42.6% ± 2.1% 18 O or 96.6 % ± 4.8% of the expected 44.1%.…”

“…Aβ(1-40) was purchased from Keck Biotechnology Center (Yale University, New Haven, CT) and purified according to the protocol described previously [18]. A 25-µM stock solution of Aβ monomer was prepared in 2 mM tris buffer (pH 7.5).…”

Probing the origins, diagnosis and treatment of amyloid diseases using antibodies

“…The determination of detailed structures of the mature fibrils is also challenging due to their intractable and frequently heterogeneous nature, which again seriously limits the application of the traditional methods of structural biology such as solution NMR spectroscopy and X-ray diffraction techniques. Structural information concerning amyloid fibrils has, however, been obtained from atomic force microscopy (AFM) [30,31], FTIR [32,33], X-ray fibre diffraction studies [17], cryoelectron microscopy [34,35], hydrogen/deuterium exchange analysed by mass spectrometry and NMR [36][37][38][39] and solid state NMR [40,41]. Important information about both the structures of the aggregates and the mechanism of their formation has also been obtained by using methods such as limited proteolysis [42,43], systematic site-directed mutagenesis [44][45][46], and the analysis of the effects of interactions with specific antibodies.…”