AMIDE HYDROGEN-EXCHANGE AND MASS-SPECTROMETRY - A PROBE OF HIGH-ORDER STRUCTURE IN PROTEINS

A new analytical method which facilitates peptide amide hydrogen as a tool for detecting conformational changes and probing high-order structure in large proteins is described. Following a period of deuterium exchange-in, the protein is placed in slow exchange conditions and fragmented into peptides with pepsin. The peptides are analyzed by directly-coupled HPLC fast atom bombardment mass spectrometry to determine their deuterium content. Results presented here demonstrate that this method can be used to determine rate constants for peptide amide hydrogen exchange, and to detect the thermal denaturing of cytochrome c.