Advancing Hydrogen Deuterium Exchange Mass Spectrometry Through the development of Novel Internal Exchange Reporters

Abstract

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis. The remarkable versatility and sensitivity of HDX-MS renders the technique uniquely well suited to probing challenging biological systems. Over the last two decades advancements in mass spectrometers, sample handling techniques, and software tools have brought HDX-MS into the mainstream by dramatically improving data quality. Despite this renaissance, HDX-MS measurements remain very challenging to reproduce. The strong solution dependence of the chemical process underlying amide HDX contributes significantly to irreproducibility; even small variations in temperature or pH can have a pronounced effect on the deuterium uptake by a protein. As a result, a myriad of controls have been developed to help the investigator identify and address variations in solution conditions. Among these controls are compounds which directly monitor the conditions under which amide exchange takes place. These compounds, referred to as internal exchange reporters (IERs), have been the focus of my graduate work in the Guttman Lab at the University of Washington. When my work in the area began, the concept of reporting controls had been well established. However, the reporters themselves, small unstructured peptides, were of limited utility. Under the guidance of Dr. Miklos Guttman and with the assistance of many brilliant and rather patient friends and colleagues I have been able to develop novel imidazolium based IERs. This document will describe the design, validation, and subsequent refinement of these compounds through the discussion of previously published and ongoing work.