Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen-Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

Hanliu Wang, Don L. Rempel, Daryl Giblin, Carl Frieden, Michael L. Gross

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions. (Figure Presented).

Original languageEnglish
Pages (from-to)10687-10695
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number20
DOIs
StatePublished - Oct 17 2017

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