Hydrogen Exchange Mass Spectrometry for the Analysis of Ligand Binding and Protein Aggregation

Abstract

Most biological processes are performed by protein-ligand complexes that act in a coordinated manner. Any interruption in protein-ligand interaction can cause failure of the whole system. Thus, it is important to understand in detail how proteins and ligands interact. Mass spectrometry (MS) has become a powerful analytical approach for proteins. Hydrogen exchange (HX) has been under development since the 1950s. When coupled with MS detection, its use has increased profoundly because it can follow protein conformational changes, dynamics, and folding with relative ease and high sensitivity. This chapter focuses on methodology and analysis for HX-based studies of protein-ligand and protein-protein interactions. There are two approaches based on HX to determine protein-ligand binding affinity: 'protein-ligand interactions by mass spectrometry, titration, and hydrogen exchange' (PLIMSTEX) and 'stability of unpurified proteins from rates of hydrogen exchange' (SUPREX). The methods are applicable when the target protein undergoes HX via an EX2 mechanism.