Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions

Abstract

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery.

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

“…A synthetic, wild-type human form of Aβ 40 (Keck Biotechnology Resource Laboratory) and a recombinant, wild-type human form of Aβ 42 (rPeptide) were used. Sample preparation followed a reported procedure (49) as detailed in Supporting Information.…”

“…More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1,2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 .…”

“…In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1,2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3)(4)(5)(6)(7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8)(9)(10)(11)(12)(13).…”

“…MS, however, was used for analyzing the aggregated Aβ fibrils (31-33) and, with ion mobility (34)(35)(36), for soluble Aβ aggregates. Hydrogen-deuterium exchange (HDX) (37)(38)(39)(40)(41)(42), even with top-down sequencing, can afford residue-level information (43,44) and provide insight on Aβ 42 fibril core structure (31,32) and its recycling (33,45).…”

“…In light of the dearth of aggregation studies at the peptide level, we have used herein pulsed HDX to study the aggregation of the Aβ 40 and Aβ 42 peptides. Our platform is suitable for confirming the effect of temperature, agitation, and presence of Cu 2+ ions on Aβ aggregation.…”