High resolution, high-throughput amide deuterium exchange-mass spectrometry (DXMS) determination of protein binding site structure and dynamics: utility in pharmaceutical design

Abstract

Mass spectrometry-based peptide amide deuterium exchange techniques have proven to be increasingly powerful tools with which protein structure and function can be studied, and are unparalleled in their ability to probe sub-molecular protein dynamics. Despite this promise, the methodology has remained labor-intensive and time consuming, with substantial limitations in comprehensiveness (the extent to which target protein sequence is covered with measurable peptide fragments) and resolution (the degree to which exchange measurements can be ascribed to particular amides). I have developed and integrated a number of improvements to these methodologies into an automated high throughput, high resolution system termed Deuterium Exchange Mass Spectrometry (DXMS). With DXMS, complete sequence coverage and single-amide (amino acid) resolution are now rapidly accomplished. DXMS is designed to work well with large proteins and when only small amounts of material are available for study. Studies can be performed upon a receptor-ligand pair as they exist on or within a living cell (in vivo) without prior purification, allowing effective in situ study of integral membrane protein receptors. We have ambitious initiatives underway to make DXMS widely available both for basic academic research studies and commercial drug discovery efforts. In this paper I present an overview of DXMS technology and highlight some of the benefits it will provide in drug discovery and basic proteomics research.