Indu Kheterpal

Enhanced correction methods for hydrogen exchange-mass spectrometric studies of amyloid fibrils

We describe methods for minimization of and correction for artifactual forward and backward exchange occurring during hydrogen exchange–mass spectrometric (HX–MS) studies of amyloid fibrils of the Aβ(1–40) peptide. The quality of the corrected data obtained using published and new correction algorithms is evaluated quantitatively. Using the new correction methods, we have determined that 20.1 ± 1.4 of the 39 backbone amide hydrogens in Aβ(1–40) exchange with deuteriums in 100 h when amyloid fibrils of this peptide are suspended in D2O. These data reinforce our previous conclusions based on uncorrected data that amyloid fibrils contain a rigid protective core structure that involves only about half of the Aβ backbone amides. The methods developed here should be of general value for HX–MS studies of amyloid fibrils and other protein aggregates.

Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of Protein Aggregates

The elucidation of the structure of amyloid fibrils and related aggregates is an important step toward understanding the pathogenesis of diseases like Alzheimers disease, which feature protein misfolding and/or aggregation. However, the large size, heterogeneous morphology, and poor solubility of amyloid-like fibrils make them resistant to high-resolution structure determination. Using amyloid fibrils and protofibrils of the Alzheimers plaque peptide amyloid beta as examples, we describe here the use of hydrogen/deuterium exchange methods in conjunction with electrospray ionization mass spectrometry to determine regions of the peptide involved in beta-sheet network when it is incorporated into protein aggregates. The advantages of this method are low sample utilization and high speed. The basic methodology exploits the fact that protons either involved in H-bonded secondary structures or buried in a proteins core structure exchange more slowly with deuterium than do solvent-exposed and non-H-bonded protons. Details of all aspects of this methodology, including sample preparation, data acquisition, and data analysis, are described. These data provide insights into the structures of monomers, protofibrils, and fibrils and to the structural relations among these states.