Conformations of Disulfide-Intact and -Reduced Lysozyme Ions Probed by Proton-Transfer Reactions at Various Temperatures

Abstract

Proton-transfer reactions of disulfide-intact and -reduced lysozyme ions (7+ through 14+) to 2,6-dimethylpyridine were examined in the gas phase using tandem mass spectrometry with electrospray ionization. By changing temperature of a collision cell from 280 to 460 K, temperature dependence of reaction rate constants and branching fractions was measured. Absolute reaction rate constants for the protein ions of specific charge states were determined from intensities of parent and product ions in the mass spectra. Remarkable change was observed for the rate constants and distribution of product ions. The rate constants for disulfide-intact ions changed more drastically with change of charge states and temperature than those for disulfide-reduced ions. Observed branching fractions for parent and product ions were represented by calculated reaction rate constants with a scheme of sequential process. The reaction rate constants are closely related to conformation changes with change of temperature, which are profoundly influenced by amputation of disulfide bonds.

Temperature-Resolved Proton Transfer Reactions of Biomolecular Ions

“…Details of the experimental apparatus used in the present study have been described elsewhere. [15][16][17] Brie y, the system consists of four vacuum chambers with an ESI source, a tandem mass spectrometer, and a gas cell equipped with an octapole ion trap. Multiply-protonated peptide and protein ions were produced by ESI.…”

“…Details of scheme used to determine absolute values of the rate constants have been described elsewhere. 16,17) In Fig. 2, the reaction rate constants for proton transfer from [Ang+2H] 2+ , to 1-Bu, dipropylamine (Dpr), 3,5-dimethylpyridine 30) (35Dmpy), and 2,6-dimethylpyridine (26Dmpy) are plotted as a function of temperature in the gas cell.…”

“…Proton transfer reactions of disul de-intact and -reduced lysozyme ions, [Lys i +zH] z+ and [Lys r +zH] z+ , respectively, (7+ through 14+) to 26Dmpy were examined. 16) e disul dereduced form of lysozyme was obtained by boiling a lysozyme in an aqueous 0.01 M dithiothreitol (DTT) solution for 30 min. 16) Time-of-ight mass spectra of the disul deintact lysozyme ions for a 11+ charge state, [Lys i +11H] 11+ reacted with 26Dmpy at various temperatures are shown in Fig.…”

“…e structures and reactions of gas-phase biomolecules have been studied extensively using various mass spectrometric methods. [8][9][10][11][12][13][14][15][16][17] Protein conformations and dynamics have been studied extensively by mass spectrometry-based approaches to characterize kinetic intermediates in refolding experiments. A new experimental approach would be expected to allow exact biomolecular structures to be determined in the gas phase.…”

“…10) Proton transfer reactions of angiotensin I, disul de-intact and -reduced lysozyme, and ubiquitin ions for multiplyprotonated states are discussed in this study. [15][16][17] e temperature dependence of the absolute reaction rate constant for proton transfer and the distribution of product ions were examined for those peptide and protein ions in the gas phase. An issue that is attracting considerable attention is their gas-phase conformations that could resemble structural evolution that originated from temperature in the gas phase.…”