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 protein conformation and folding and to the study of protein-ligand interactions and requires no modifications of the instrument. Amide protons were allowed to exchange with deuterons in buffered D2O at room temperature, pD 7.25. Exchanged deuterons were "frozen" in the exchanged state by quenching at pH 2.5, 0 degree C and analyzed by MALDI-TOF MS. The matrix mixture consisted of 5 mg/mL alpha-cyano-4-hydroxycinnamic acid, acetonitrile, ethanol, and 0.1% TFA. The matrix was adjusted to pH 2.5, and the chilled MALDI target was rapidly dried. Deuteration of amide protons on cyclic AMP-dependent protein kinase was measured after short times of incubation in deuterium by pepsin protein digestion and MALDI-TOF MS analysis. The unseparated peptic digest was analyzed in a single spectrum of the mixture. From five spectra, H/D exchange rates were determined for some 40 peptides covering 65% of the protein sequence.

The transferrins are a class of iron-binding proteins that require the presence of a synergistic anion for conformation-dependent binding of ferric ions. Bromopyruvate, a known synergistic anion and affinity label of ovotransferrin (oTF) [Bailey, C. T., Patch, M. G., & Carrano, C. J. (1988) Biochemistry 27, 6276-6282], was used to probe the structure of the metal- and anion-binding sites of the functional N- and C-terminal proteolytic halves (oTF/2N and oTF/2C, respectively) of ovotransferrin. Incubation of oTF/2N with [2-14C]bromopyruvate in the presence of Fe3+ ions resulted in the incorporation of 0.70 mol of 14C label/mol of oTF/2N; 14C-labeled oTF/2N was then purified and digested sequentially with trypsin and V8 protease to determine the sites of modification. Quantification of 14C radioactivity, analysis of purified 14C-labeled peptides by gas-phase sequencing and mass spectrometry demonstrated that chemical modification was restricted to nucleophilic residues contained in a fragment corresponding to residues 189-204 of oTF/2N, including Lys 199, Lys 202, and His 196. Lysine 199 was also protected from modification with [3H]CH2O in iron-saturated oTF/2N, suggesting the involvement of this residue in anion binding by the apo conformation [Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V., & Baker, E. N. (1990) Nature 344, 784-787]. Lysine 199 is conserved as a basic residue in the N-terminal metal-binding domains of the transferrins but not in the homologous C-terminal metal-binding domains. Identical trials with oTF/2C showed binding, but not modification, with bromopyruvate. These data suggest that Lys 199, Lys 202 and His 196, which are located on an alpha-helix (8) that terminates at the anion-binding site [Dewan, J. C., Mikame, B. Hirose, M., & Sacchettini (1993) Biochemistry 32, 11963-11968], attract and channel the synergistic anion to the anion-binding site. The presence or absence of basic residues in the metal-binding lobes of transferrins may account for the different anion- and metal-binding characteristics observed for the iron-binding sites of these proteins.