Folding analysis of hormonal polypeptide calcitonins and the oxidized calcitonins using electrospray ionization mass spectrometry combined with H/D exchange

Abstract

Conformational change of calcitonins has been examined by measuring the rate of hydrogen/ deuterium (H/D) exchange in amino acids. Time dependent m/z shift caused by H/D exchange was monitored by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT MS). The rate constants of H/D exchange were calculated from apparent first-order kinetics. The time course of H/D exchange exhibited two phases of faster and slower exchange. The smaller rate constant (k 2 ) estimated from the slower H/D exchange was correlated with an ␣-helix content that reflected the folding state. The order of k 2 values obtained for human calcitonin (hCT), porcine calcitonin (pCT), salmon calcitonin (sCT), and elcatonin (ECT) was hCT Ͼ pCT Ϸ sCT Ͼ ECT. Although the amino acid sequence of sCT is similar to that of ECT, their k 2 values were considerably different. The results suggest that ECT is relatively rigid on the N-terminal side cyclic structure in the folded state. Further, the effect of methionine oxidation on k 2 has been examined. In the oxidized pCT that possesses similar biological activity with the intact pCT, the k 2 values obtained were nearly equal. The k 2 of hCT increased via methionine oxidation, and the biological activity was weakened by oxidation. This suggested that methionine oxidation of hCT produced unfolding in the secondary structure and that oxidative unfolding of hCT led to the loss of biological activity. The results indicate that the H/D exchange rate constant may be used as an informative parameter to elucidate the relationship between the folded state and biological activity of polypeptides like calcitonins with secondary structure. (J Am Soc Mass Spectrom 2004, 15, 1556 -1564

The effect of alkylamine additives on the sensitivity of detection for paclitaxel and docetaxel and analysis in plasma of paclitaxel by liquid chromatography–tandem mass spectrometry

“…The binding sites for the alkylamine ion were investigated by evaluation of amine adduct ion formation for analogs of paclitaxel. Hydrogen/deuterium (H/ D) exchange combined with mass spectrometry is a valuable tool to study protein-ligand interaction and protein-protein interactions (Nabuchi et al, 2004;Zhu et al, 2004). Recently, Lorenz and co-workers applied this approach to demonstrate the non-covalent dimerization of paclitaxel in solution and to investigate a drug-drug complex as being formed in solution or in the gas phase (Lorenz et al, 2001(Lorenz et al, , 2002.…”

Ion Mobility and Collision-Induced Dissociation Analysis of Carbonic Anhydrase 2

“…Though the sample solution passes through the electrospray needle which has a high electric potential, analytes are kept in solution until the final ionization step. For the analysis of protein folding, one of the established methods is to measure the hydrogen/deuterium exchange. − In addition, the shift in charge state distribution of multiply protonated proteins, [M + n H] n + , allows monitoring of different conformations of the analytes. For example, a conformation corresponding to an unfolded protein will lead to the formation of higher charge state distributions, whereas a folded protein leads to lower charge states.…”

Nanosecond Laser-Induced Photochemical Oxidation Method for Protein Surface Mapping with Mass Spectrometry

“…Oxidation by OH radical is a random process dependent on the solvent-accessible surface as well as the chemical properties of the exposed amino acids . Upon oxidation, the three-dimensional structure of a protein may change to a certain extent, depending on the sites of oxidation . As a result, any further oxidations after this conformational change can result in erroneous allocation of surface-accessible residues.…”