MEASUREMENT OF AMIDE HYDROGEN-EXCHANGE BY MALDI-TOF MASS-SPECTROMETRY

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was used to determine amide proton/deuteron (H /D) exchange rates, The method has broad application to the study of p rotein conformation and folding and to the study of protein-ligand int eractions and requires no modifications of the instrument. Amide proto ns were allowed to exchange with deuterons in buffered D2O at room tem perature, pD 7.25, Exchanged deuterons were ''frozen'' in the exchange d state by quenching at pH 2.5, 0 degrees C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinna mic acid, acetonitrile, ethanol, and 0.1% TFA, The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteratio n of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digest ion and MALDI-TOF MS analysis. The unseparated peptic digest was analy zed in a single spectrum of the mixture. From five spectra, H/D exchan ge rates were determined for some 40 peptides covering 65% of the prot ein sequence.