ABSTRACT
The advent of mass spectrometers capable of analyzing proteins from conditions under which the protein remains close to its native state initiated studies of higher order protein structure. These studies have included both protein-protein and protein-ligand interactions as well studies of the relative stability of protein variants. To observe these interactions and stability differences using mass spectrometry (MS) it is necessary to preserve higher order protein structure in the gas phase. The higher order structure of a protein is defined as the spatial arrangement of amino acid residues into elements of secondary structure such as a-helices and {3sheets. These elements of secondary structure arise from local hydrogen bonding interactions, while the spatial interactions of amino acids, far apart in the linear sequence, impose conformations on the protein and give rise to the tertiary structure. For application of MS to higher order protein structure a method is required in which differences in conformations of proteins may be monitored. ln the early days of electrospray ionization (ESI) MS, many studies reported changes in the charge state distribution arising from different conformations of the protein. These changes in charge state distribution are observed for proteins in the presence and absence of cofactors or ligands. For example, Fig. I shows the ESI mass spectra obtained from bovine a-lactalbumin in the presence and absence of Ca2 +. The protein with Ca2 + present shows the expected increase in mass and in addition a lower charge state distribution. However, comparison of charge states to show differences in tertiary structure relies on keeping all instrument parameters and solution conditions identical. In particular, changes in solvent composition, often necessary to effect changes in protein conformation (discussed later), also affect the charge state distribution fl]. While it is possible to use changes in charge state distribution as a "fingerprint" for studying three-dimensional (3D) structure of proteins, a more reliable method is required. One such method, successfully applied in nuclear magnetic resonance (NMR). is that of hydrogen deuterium exchange labeling (HX) [2].
