Improved methods for high-resolution identification of solvent-accessible amide hydrogens in polypeptides and for characterization of polypeptide structure

摘要

The present invention provides methods of determining, at a resolution of about 1-5 amino acid residues, the position of a peptide amide hydrogen that has been labeled with an isotope of hydrogen other than 1H by determining the quantity of isotope and/or rate of exchange of peptide amide hydrogen(s) with isotope. Invention methods comprise generating a population of sequence-overlapping endopeptidase fragments of the labeled protein under conditions of slowed hydrogen exchange and then deconvoluting fragmentation data. Invention methods allow for the localization of labeled peptide amide positions in an amino acid-specific manner, thereby providing information on residues involved in binding sites, surface conformation and accessibility of residues, and conformational changes at different times or conditions, for example.