Probing protein ligand interactions by automated hydrogen/deuterium exchange mass spectrometry.
Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARgamma induced upon binding a full agonist and a partial agonist. PPARgamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARgamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARgamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARgamma/RXRalpha LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.
Scripps Research Institute, 5353 Parkside Drive (RF-1), Jupiter, FL 33458, USA.
Michael J
MJ
Chalmers
Scott A
SA
Busby
Bruce D
BD
Pascal
Yuanjun
Y
He
Christopher L
CL
Hendrickson
Alan G
AG
Marshall
Patrick R
PR
Griffin
D000595:Amino Acid Sequence; D001331:Automation; D003903:Deuterium; D005583:Fourier Analysis; D006859:Hydrogen; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*
D008024:Ligands; D015335:Molecular Probes; D011506:Proteins; D006859:Hydrogen; D003903:Deuterium