Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions.
The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.
Ehring
H
H
Department of Structural Chemistry, Pharmacia & Upjohn, Stockholm, S-112 87, Sweden. Hanno.Ehring@pyrosequencing.com
D000595:Amino Acid Sequence; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000737:chemistry* / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry
D018970:Insulin-Like Growth Factor Binding Protein 1; D010446:Peptide Fragments; D007334:Insulin-Like Growth Factor I