Conformations of Disulfide-Intact and-Reduced Lysozyme Ions Probed

Article pubs.acs.org/JPCB

Conformations of Disulfide-Intact and -Reduced Lysozyme Ions Probed by Proton-Transfer Reactions at Various Temperatures Shinji Nonose,* Kazuki Yamashita, Takuya Okamura, Satoshi Fukase, Minami Kawashima, Ayako Sudo, and Hideo Isono Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan S Supporting Information *

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.

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INTRODUCTION Investigations of gas-phase protonated biomolecules are motivated by the growing prevalence of electrospray ionization (ESI) mass spectrometry. By means of development of ESI, it is now possible to place ions of large biomolecules into the gas phase without any destruction.1,2 With combination of ESI, mass spectrometry could be applied to analysis of large biological molecular systems. In addition to applications in bioanalytical chemistry, access to biomolecules in the gas phase provides an ideal opportunity to study them in isolated states. The advantage of gas-phase studies is that ideas can be tested on simple model systems.3−5 Substantial progress has been made in the past 40 years toward achieving this understanding for simple reactions of small molecular systems through experimental developments.6−8 By contrast, protein folding is so complex that the connection between the sequence and three-dimensional structure of a protein, and the mechanism of the folding process itself, are not understood despite many years of study.9 The identification and characterization of intermediates provide important insights into the mechanism of folding. Protein conformations and dynamics have been studied extensively by mass-spectrometry-based approaches to characterize kinetic intermediates in refolding experiments.10,11 By introducing a biological molecule into the gas phase, it is possible to separate its hydration interactions and intramolecular interactions and examine them independently. Structures and reactions of gas-phase biomolecules have been studied extensively using various mass spectrometric methods.12−19 Ion mobility mass spectrometry has been studied for © 2014 American Chemical Society

various protein ions to examine their gas-phase conformations.20−24 Proton-transfer reactions of multiply protonated protein and peptide ions have been examined.25−29 Protontransfer reactions involve H/D exchange.30−34 Regarding combining proton-transfer reactions and collision-induced dissociation (CID), charge manipulation of multiply charged protein and peptide ions prior and post CID has been studied.35−40 A new experimental approach is expected to determine exact biomolecular structures in the gas phase.41 Lysozyme is a relatively small protein (14 307 Da for lysozyme from chicken egg white) which is composed of 129 residues of amino acids. It contains 18 basic residues (11 arginines, 6 lysines, 1 histidine, and the N-terminal amino group). It has four intramolecular disulfide bonds at cysteine residues.42 Since its discovery in 1922 by Alexander Fleming,43 this protein has represented one of the most popular and important model systems for understanding the complexity of protein structure and function. Lysozyme and its derivative of disulfide linkages in solution phase has been investigated by a wide range of complementary biophysical techniques.44−46 The role of disulfide bridges in the formation and maintenance of the three-dimensional fold of lysozyme has been studied. Formations of fibrils by lysozyme in the presence and absence of its native disulfide bonds have been also examined.47,48 It has been found that they profoundly influence the fibrillar Received: June 6, 2014 Revised: July 11, 2014 Published: July 21, 2014 9651

dx.doi.org/10.1021/jp505621f | J. Phys. Chem. B 2014, 118, 9651−9661

The Journal of Physical Chemistry B

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Chemicals) in a 1:30 water:methanol mixture which also contained 0.1−1.0% v/v acetic acid. The disulfide-reduced form of lysozyme was obtained by boiling a lysozyme in 0.01 M dithiothreitol (DTT) aqueous solution for 30 min.49,54,57,61 The resulting solution was diluted with methanol and acetic acid such that the electrosprayed solution was ∼1:30 water:methanol with 0.1−1.0% v/v acetic acid. Mass spectra of disulfide-intact and disulfide-reduced lysozyme ions were also measured by means of a commercial ESI-QIT mass spectrometer (Bruker Daltonics, HCT-ultra ETD II). Before the measurements, the mass range of the mass spectrometer was calibrated with a chemical standard for mass calibration (Agilent Technologies, ESI Tuning Mix). Mass deconvolution results of the mass spectra show an 8.6 ± 0.5 Da increase in mass for reduced lysozyme compared with the native protein. If all four of the disulfide bonds were reduced, molecular weight is expected to increase by 8 Da. Though the value of mass deconvolution results was slightly larger than an expected value, it would demonstrate that all four of the disulfide bonds were reduced.

morphology and cytotoxicity. Namely, disulfide bonds stabilize folded conformations. There are a number of gas-phase studies for multiply protonated lysozyme ions,49−72 which include H/ D exchange, 5 0 − 5 2 field asymmetric waveform IMS (FAIMS),53,54 and radical-directed dissociation.55,56 In particular, ion mobility mass spectrometry has been studied for multiply protonated lysozyme ions.57−60 It has been shown that conformations in the gas phase are highly dependent on the presence of disulfide bonds. Conformations of the disulfideintact protein ions are highly folded, whereas disulfide-reduced protein ions favor highly diffuse, unfolded forms of the protein, since there are no disulfide bonds to impose structural restrictions.57 Proton-transfer reactions of lysozyme ions have been studied by Williams and co-workers.61 It has been reported that reaction rate constants are closely related to the presence of disulfide bonds. The proton-transfer reactions between a small molecule and a small molecular ion are known as a typical elementary process for ion−molecule reaction.7,8 Proton-transfer reactions of peptide and protein ions to primary, secondary and aromatic amines, such as pyridine, 2methylpyridine, and 2,6-dimethylpyridine (Dmpy), have been examined,28,29 and it has been found that steric hindrance of Dmpy is relatively not so large. In the present study, disulfideintact and -reduced lysozyme ions for multiply protonated state, [M + zH]z+, were produced by using ESI. Proton transfer from [M + zH]z+ to Dmpy was investigated in the gas phase. Absolute reaction rate constant for proton transfer was determined. Temperature dependence of reaction rate constant and distribution of product ions were examined. On the basis of experimental observations, we discussed conformation change of lysozyme ions with and without disulfide bonds as a function of temperature. Relationship between gas-phase conformation and disulfide linkage was also discussed.

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RESULTS AND DISCUSSION Mass Spectra and Branching Ratios of DisulfideReduced and -Reduced Lysozyme Ions Reacted with 2,6-Dimethylpyridine. Typical time-of-flight mass spectra of disulfide-intact lysozyme ions are presented in Figure 1 as a

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EXPERIMENTAL METHODS Details of the experimental apparatus used in the present study are described in the Supporting Information. Briefly, 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 disulfide-intact lysozyme ions, [Mi + zH]z+, and disulfide-reduced lysozyme ions, [Mr + zH]z+, were produced by ESI of a dilute solution of lysozyme from egg white in a methanol−water mixture including acetic acid. The ions produced by ESI were admitted into the vacuum through a stainless capillary. A specific charge state of ions was selected by a quadrupole mass spectrometer (QMASS). The chargeselected lysozyme ions emerging from QMASS were admitted into the gas cell equipped with an octapole ion trap. The gas cell was filled with He including gaseous molecules. Dmpy was chosen as a target molecule to collide with ions and to induce proton-transfer reactions. Temperature dependence of reaction rate constants and branching fractions for proton transfer from charge-selected lysozyme ions to the target molecules was measured, by changing temperature of the gas cell from 280 to 460 K. Temperature of the gas cell was continuously monitored. Parent and product ions were extracted from the gas cell and were mass-analyzed by a time-of-flight mass spectrometer equipped with reflectron (TOFMS). The ESI charge state distributions for lysozyme depend on the properties of the solution. Details of electrospraying disulfide-intact and disulfide-reduced lysozyme solutions are described in Supporting Information. Briefly, the disulfideintact solution was 5 × 10−5 M of lysozyme (egg white, Wako

Figure 1. Time-of-flight mass spectra of [Mi + 11H]11+ reacted with Dmpy at various temperature. (A) Mass spectrum of all ions produced with ESI, where dc voltage in QMASS was put off. (B) Ions of specific charge state, [Mi + 11H]11+, were selected with QMASS as a reactant. (C−G) Mass spectra of [Mi + 11H]11+ reacted with Dmpy at various temperatures. Temperature of gas cell was (C) 460 K, (D) 410 K, (E) 360 K, (F) 310 K, and (G) 290 K. 9652



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Figure 2. Observed and reappeared branching fractions of parent ion, [Mi + 11H]11+, and product ions, [Mi + z′H]z′+ (z′ = 7−10), plotted as a function of temperature in the gas cell. (a) Observed branching fractions of parent ion and product ions. (b) Reappeared branching fractions. Details of the representation scheme are described in the text.

shown in these figures, a protonated molecular ion, Dmpy·H+, and a protonated dimer ion, (Dmpy)2·H+, were also detected. Following these experimental observations, it was confirmed that proton transfer from lysozyme ions, [M + zH]z+, to Dmpy proceeds in the gas cell

function of mass-to-charge ratio (m/z). In Figure 1A, a mass spectrum of multiply protonated lysozyme ions, [Mi + zH]z+ (z = 6−12), is presented. By putting off dc voltage in QMASS, all ions, which were produced with ESI, transmitted through QMASS. In Figure 1B, ions of specific charge states, [Mi + 11H]11+, were selected as reactant with QMASS. Typical mass resolution of QMASS, m/Δm, was ∼100. During adjustments of rf amplitude and dc voltage in QMASS, mass spectra were measured by TOFMS to monitor charge selection with QMASS. It was confirmed that the mass resolution was good enough to select specific charge states of ions. In this spectrum, a small amount of product ions, [Mi + z′H]z′+ (z′ = 10), was observed. These product ions were generated from release of protons in the gas cell. For the mass spectrum, temperature of the gas cell was ∼460 K. Gas-phase basicity (GB) of [Mi + 11H]11+ has been reported to be 841 kJ/mol.61 These product ions were not generated from unimolecular release of protons by thermal radiation. Collision with He would induce the release. Temperature dependence of these decompositions was measured for ions of various charge states. Release yield decreased monotonically with decrease of temperature and of charge state. In Figure 1C−G, mass spectra of [Mi + 11H]11+ reacted with Dmpy at various temperatures are presented. As a target molecule, Dmpy was introduced into the gas cell, as well as He. Temperature of gas cell was (C) 460 K, (D) 410 K, (E) 360 K, (F) 310 K, and (G) 290 K. As shown in the figures, a lot of product ions, [Mi + z′H]z′+ (z′ = 7−10), were observed. Not

[M + z H]z + + Dmpy → [M + (z − 1)H](z − 1) + + Dmpy ·H+

(1)

Proton affinity (PA) of Dmpy is 963.0 kJ/mol, and is smaller than that of isolated basic amino acids, such as lysine, histidine, and arginine.73 By Coulomb repulsion between charges in [M + zH]z+, intrinsic proton affinities of [M + zH]z+ become lower than that of Dmpy, and the proton is able to transfer from [M + zH]z+ to Dmpy by collision. The reaction is an exoergic process. Because the concentration of ionic species such as [M + (z −1)H](z−1)+ and Dmpy·H+ in the cell is much smaller than that of neutral Dmpy, the reverse reaction, [M + (z − 1)H](z−1)+ + Dmpy·H+ → [M + zH]z+ + Dmpy, does not occur even if it is an exoergic or exothermic process. Integrated intensities of peaks in the mass spectra were estimated. Normalized branching fractions of ions are defined as Bz ′ =

Iz ′ ∑ Iz ″ z ″≤ z

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(2)



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z′H]z′+ (z′ = 10−13), were observed. In Figure 4a, the observed branching fractions of the parent ion, [Mr + 14H]14+, and product ions, [Mr + z′H]z′+ (z′ = 10−13), are plotted as a function of temperature in the gas cell. Abundance of the parent ion, [Mr + 14H]14+, decreased monotonically with decrease of temperature. That of the product ion, [Mr + 13′H]13′+, increased gradually from 460 to 400 K with decrease of temperature, whereas it decreased below 400 K. On the other hand, that of [Mr + 12′H]12′+ increased from 460 to 360 K, whereas it almost kept constant below 360 K. Abundance of [Mr + 11′H]11′+ increased monotonically with decrease of temperature. Abundance of [Mr + 10′H]10′+ increased from 460 to 295 K with decrease of temperature, whereas it decreased below 295 K. As described above, dramatic changes of fractions were observed for each ion. The changes of fractions would relate with remarkable changes of reaction rate constant. Absolute reaction rate constants for proton transfer were estimated. We represented observed branching fractions, by reaction rate constant of each ion, based on assumption of sequential process. These results are presented in Figures 2b and 4b. Details of the reappearance scheme are described in a later section. Reaction Rate Constants of Proton Transfer from Disulfide-Intact and -Reduced Lysozyme Ions to 2,6Dimethylpyridine. Following eq 1, absolute reaction rate constants, k, of proton transfer from [M + zH]z+ to gaseous target molecules (Dmpy) were determined. Details of the scheme have been described elsewhere.28 The reaction rate constant, k, is given as

where Iz′ is the integrated intensity of peaks for parent and product ions and Bz′ is the observed branching fraction for the ion. Peak intensities for Dmpy·H+ and (Dmpy)2·H+ were not taken into account in the estimation of branching fractions by eq 2. In Figure 2a, the observed branching fractions of parent ion, [Mi + 11H]11+, and product ions, [Mi + z′H]z′+ (z′ = 7− 10), are plotted as a function of temperature in the gas cell. Abundance for each ion changed dramatically with temperature change. Abundance of the parent ion, [Mi + 11H]11+, decreased monotonically with decrease of temperature. That of the product ion, [Mi + 10′H]10′+, decreased from 460 to 350 K with decrease of temperature, whereas it increased below 350 K. That of [Mi + 9′H]9′+ decreased from 460 to 370 K, whereas it increased below 370 K. On the other hand, that of [Mi + 8′H]8′+ increased from 460 to 370 K, whereas it decreased below 370 K. That of [Mi + 7′H]7′+ increased gradually from 460 to 350 K, whereas it decreased below 350 K. Time-of-flight mass spectra of disulfide-reduced lysozyme ions are presented in Figure 3 as a function of mass-to-charge

k=

⎛ [(M + z H)z + ]0 ⎞ 1 ln⎜ ⎟ [Dmpy]t ⎝ [(M + z H)z + ] ⎠

(3)

where [Dmpy] is number of Dmpy in unit volume, t is reaction time, and [(M + zH)z+] is density of parent ion, [M + zH]z+. [(M + zH)z+]0 is initial density of parent ion before reaction, which is assumed to be equal to summation of intensity for parent and all product ions in the mass spectra. [(M + zH)z+]0 almost keeps constant during the measurement over temperature range. The temperature-dependent mass spectra were measured several times. It was confirmed that reappearance in the intensity ratio was good enough. [Dmpy] is assumed to be in significant excess of [(M + zH)z+]. [Dmpy] keeps constant during the measurement. As shown in Figures 1B and 3B, small amounts of product ions by proton release were observed. In order to obtain intrinsic values of [(M + zH)z+]0/[(M + zH)z+], the ratio of the proton release was considered to compensate intensities. In order to obtain the exact value of [Dmpy] in the gas cell, pressure in the third vacuum chamber was measured with ionization vacuum gauge. Detection efficiency for Dmpy with ionization vacuum gauge is assumed to be equal to that for n-heptane. This value is 7.6 compared with that of N2.74−76 Conductance of the gas cell and the third vacuum chamber was estimated by considering the evacuation rate of the diffusion pump and diameter of entrance and exit hole of the gas cell. It is confirmed that the pressure in the gas cell is 172 ± 5 times higher than that in the third vacuum chamber. For protonated ions, Dmpy·H+, which is generated by proton transfer from [M + zH]z+, subsequent clustering reaction occurs such as, Dmpy·H+ + Dmpy → (Dmpy)2·H+. The reversible reaction, Dmpy·H+ + Dmpy ← (Dmpy)2·H+, was negligible at low temperature (T ≈ 290 K), and the dimer

Figure 3. Time-of-flight mass spectra of [Mr + 14H]14+ reacted with Dmpy at various temperature. (A) Mass spectrum of all ions produced with ESI, where dc voltage in QMASS was put off. (B) Ions of specific charge state, [Mr + 14H]14+, were selected with QMASS as a reactant. (C−G) Mass spectra of [Mr + 14H]14+ reacted with Dmpy at various temperatures. Temperature of the gas cell was (C) 460 K, (D) 410 K, (E) 360 K, (F) 310 K, and (G) 290 K.

ratio (m/z). In Figure 3A, a mass spectrum of multiply protonated lysozyme ions, [Mr + zH]z+ (z = 8−17), is presented. In Figure 3B, ions of specific charge states, [Mr + 14H]14+, were selected as a reactant with QMASS. In Figure 3C−G, mass spectra of [Mr + 14H]14+ reacted with Dmpy at various temperatures are presented. The temperature of the gas cell was (C) 460 K, (D) 410 K, (E) 360 K, (F) 310 K, and (G) 290 K. As shown in the figure, a lot of product ions, [Mr + 9654



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Figure 4. Observed and reappeared branching fractions of parent ion, [Mr + 14H]14+, and product ions, [Mr + z′H]z′+ (z′ = 10−13), plotted as a function of temperature in the gas cell. (a) Observed branching fractions of parent ion and product ions. (b) Reappeared branching fractions.

ion, (Dmpy)2·H+, was observed remarkably. A ratio, α, is defined as ⎛ [Dmpy·H+] + [(Dmpy)2 · H+] ⎞ α ≡ ln⎜ ⎟ [Dmpy·H+] ⎠ ⎝

Therefore, the relation between reaction rate constant, k, and intensity of parent ion is given as [(M + z H)z + ]0 k[Dmpy]t = [(M + z H)z + ] 1 − exp( −k[Dmpy]t )

(4)

where [Dmpy·H+] and [(Dmpy)2·H+] is intensity of Dmpy·H+ and (Dmpy)2·H+ in the mass spectra, respectively. Mass spectra of Dmpy·H+ and (Dmpy)2·H+ were measured at T ≈ 290 K every day to obtain the value of α. Frequently, [Dmpy] in the third vacuum chamber was measured with an ionization vacuum gauge. The pressure was compared with α. It was confirmed that the pressure was proportional to α. By changing the pressure more than 10 times of magnitude, it was found that uncertainties in this linear relationship between the pressure and α was at most 5%.Therefore, the [Dmpy] value was estimated from α for each measurement. For example, at measurement of mass spectra shown in Figure 1C−G, the value α was (1.3 ± 0.07) × 10−1, and [Dmpy] was (4.9 ± 0.2) × 1011 molecule cm−3. Lysozyme ions were produced continuously at ESI and were transmitted into the gas cell continuously, whereas they were extracted out as pulses to be synchronized with TOFMS. Therefore, retention time, t, of the ions trapped in the gas cell was all different. Maximum value of the retention time was 100 ms, whereas minimum value was 0. The measured values in these experiments were averaged features of various retention times. Equation 3 should be integrated with t.

(5)

The rate constant, k, is obtained as an inverse function of eq 5 from experimental value, [(M + zH)z+]0/[(M + zH)z+]. For approximation, a function expanded with power series is used. By means of scheme explained above, absolute reaction rate constants for proton transfer from [Mi + zH]z+ and [Mr + zH]z+ to Dmpy were determined. For example, the reaction rate constants at 300 K for [Mi + 11H]11+ and [Mi + 8H]8+ with Dmpy were (5.5 ± 0.3) × 10−10 and (2.3 ± 0.3) × 10−12 molecule−1 cm3 s−1, respectively. There are a lot of works to determine absolute reaction rate constants for proton transfer between small molecules and small molecular ions.7,8 For example, by means of pulsed ion cyclotron resonance spectroscopy, absolute reaction rate of proton transfer from protonated pyridine to Dmpy has been measured to be 6.0 × 10−10 molecule−1 cm3 s−1.77 Williams’ group has studied proton-transfer reactions of various peptide and protein ions. For example, the reaction rate constants for [Mi + 9H]9+ and [Mr + 9H]9+ with dipropylamine have been reported to be 5.5 × 10−12 and 1.7 × 10−12 molecule−1 cm3 s−1, respectively.61 The reaction rate constants for 7+ and 12+ charge states of cytochrome c ions with pyridine have been reported to be 1.0 and 65 × 10−12 molecule−1 cm3 s−1, respectively.25 The values 9655



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for lower charge states are smaller than that of small molecular systems. However, they have shown that the rate constants increase drastically with increase of charge state. For small molecular ions, the rate constant has been successfully interpreted by means of semiclassical collision theory.8 Reaction mechanisms of lower charge states for protein ions are seemed to be completely different from those for small molecular ions. In Figures 5 and 6, the reaction rate constants for disulfideintact and -reduced lysozyme ions, [Mi + zH]z+ (z = 7−12),

Figure 6. Absolute reaction rate constants of proton transfer for [Mr + zH]z+ (z = 7−14) are plotted as a function of temperature in the gas cell.

favor partially folded structures, whereas higher charge states (z = 10−18) of [Mr + zH]z+ adopt fully elongated conformations.57−60,62 It has also been reported that there are plural conformers in middle charge states (z = 8−10) of [Mi + zH]z+ and [Mr + zH]z+.57,62 It has also been reported that elongated conformations are formed through collisions with He.57 Variable-temperature IMS of [Mi + zH]z+ has been examined.59 Collisional heating would correspond to elevating the temperature of the gas cell. As shown in Figure 5, for [Mi + 7H]7+, the rate constant increased with decrease of temperature from 460 to 420 K, and decreased below 420 K. This would be related to structural change from partially unfolded to highly folded conformations with decrease of temperature. For [Mi + 8H]8+, the rate constant monotonically decreased with decrease of temperature. Generally, the rate constants grew larger with increase of charge states. However, exceptionally, the rate constant of [Mi + 7H]7+ was much larger than that of [Mi + 8H]8+ over all temperature range. This peculiar feature cannot be explained by results of IMS works. IMS cross sections of both ions are similar, and whole structures of them are highly folded conformations. For [Mi + 9H]9+, the rate constant increased with decrease of temperature from 460 to 360 K, and decreased below 360 K. For [Mi + 10H]10+, it increased with decrease of temperature from 460 to 350 K, and decreased below 350 K. These dramatic changes would be related to conformation change. IMS work indicates that [Mi + 9H]9+ and [Mi + 10H]10+ favor highly folded structures, and that more elongated conformations are formed with collisional heating.57 Even

Figure 5. Absolute reaction rate constants of proton transfer for [Mi + zH]z+ (z = 7−12) are plotted as a function of temperature in the gas cell.

and [Mr + zH]z+ (z = 7−14), are plotted as a function of temperature in the gas cell, respectively. We repeated the measurements several times to estimate uncertainties of the rate constants. The reaction rate constants for proton transfer are influenced by charge states of the protein ions, temperature of the gas cell, and the disulfide linkage. Protein conformations are related to Coulomb repulsion between charges, intramolecular hydrogen bonds, and self-solvation of protons by hydrophilic sites. Dramatic features of the rate constants are shown in Figures 5 and 6. If those line shapes were simple straight lines, activation barrier for reaction could be determined with Arrhenius plot. However, they are complicated curved lines, and it is impossible to estimate it. These complex features would be related to conformation change. Extended protein conformation would be entropically favorable at high temperature, whereas compact conformation would be energetically favorable at low temperature. IMS works indicate that lower charge states (z = 5−8) of [Mi + zH]z+ favor compact structures, middle charge states (z = 9−11) of [Mi + zH]z+ 9656



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Figure 7. Reaction rate constants for [Mi + zH]z+ and [Mr + zH]z+ plotted as a function of temperature in the gas cell, for comparing the same charge states. The rate constants for 7+, 8+, 9+, 10+, 11+, and 12+ charge states are presented in (a), (b), (c), (d), (e), and (f), respectively. In the figures, the letter “I” indicates the rate constants of [Mi + zH]z+, whereas the letter “R” indicates those of [Mr + zH]z+.

“R” indicates those of [Mr + zH]z+. As shown in the figure, for 9+, 10+, 11+, and 12+ charge states, the rate constants of [Mi + zH]z+ were larger than those of [Mr + zH]z+. At high temperature range, the rate constant of [Mi + 7H]7+ was equal to that of [Mr + 7H]7+, while it was smaller than that at low temperature range. On the other hand, the rate constant of [Mi + 8H]8+ was much smaller than that of [Mr + 8H]8+. For the 9+ and 10+ charge states, there were humps in the line shapes of [Mi + zH]z+, whereas the line shapes of [Mr + zH]z+ were smoothly curved lines. As a whole, the rate constants for [Mi + zH]z+ changed more drastically than those for [Mi + zH]z+ with change of temperature and charge state. In H− transfer reactions of C2H5+, s-C3H7+, t-C4H9+, and tC5H11+ with C4−C8 tertiary hydrocarbons, large negative temperature dependences of the rate constants were observed.78−80 Transition-state theory considerations relate the negative temperature dependences of these reactions to entropy changes. It is involved in the formation of the tight transition complexes as reaction intermediates. The negative temperature dependences were also found in ion−molecule association reactions.81,82 On this basis, proton transfer from highly charged lysozyme ions that are unfolded at high temperatures requires folding to transfer a proton, which would create an entropy barrier, and it exhibits negative temperature dependence. However, when they are folded at lower temperatures, proton transfer would have an enthalpy barrier or activation energy, and it exhibits positive temperature dependence. In our previous works, we have observed that reaction rate constant of proton transfer from 7+ charge state of ubiquitin ion to 1-butylamine suddenly increases with decrease of

though the compact conformations of [Mi + 9H]9+ and [Mi + 10H]10+ are most stable at lower temperature range, it could be partially unfolded at higher temperature range. At high temperature range, the rate constants of [Mi + 10H]10+, [Mi + 11H]11+, and [Mi + 12H]12+ were almost same values. For [Mi + 11H]11+ and [Mi + 12H]12+, the rate constants monotonically increased with decrease of temperature. Their structures would be partially unfolded conformations at all temperature range. As shown in Figure 6, for [Mr + 7H]7+, the rate constant increased with decrease of temperature from 460 to 370 K, and decreased below 370 K. This slight change would be related to structural change. The rate constant for [Mr + 7H]7+ was slightly larger than those for [Mr + 8H]8+ at 340−390 K. For [Mr + 8H]8+, the rate constant monotonically decreased with decrease of temperature. At high temperature range, the rate constant for [Mr + 8H]8+ was larger than those for [Mr + 9H]9+ and [Mr + 10H]10+. For [Mr + 9H]9+, it increased gradually with decrease of temperature from 460 to 370 K, and decreased gradually below 370 K. For [Mr + 10H]10+, it increased with decrease of temperature from 460 to 310 K, and decreased below 310 K. For ions of higher charge states, z = 11−14, the reaction rate constants monotonically increased with decrease of temperature. These features would be related to their fully elongated conformations at all temperature range. In Figure 7, the rate constants for [Mi + zH]z+ and [Mr + zH]z+ are plotted as a function of temperature, for comparing the same charge states. The rate constants for 7+, 8+, 9+, 10+, 11+, and 12+ charge states are presented in parts a, b, c, d, e, and f of Figure 7, respectively. In the figure, the letter “I” indicates the rate constants of [Mi + zH]z+, whereas the letter 9657



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⎧ d[Pz ] = k1[Pz ] ⎪− ⎪ dt ⎪ d[P ] ⎪ z − 1 = k1[Pz ] − k 2[Pz − 1] ⎪ dt ⎪ ⎪ d[Pz − 2] ⎨ = k 2[Pz − 1] − k 3[Pz − 2] ⎪ dt ⎪ ⎪ d[Pz ‐ 3] = k [P ] − k [P ] 3 z−2 4 z−3 ⎪ dt ⎪ ⎪ d[Pz − 4 ] = k [P ] ⎪ 4 z−3 ⎩ dt

temperature from 330 to 300 K,29 and that the rate constants for 2+ charge state of angiotensin I ion with primary amines, pyridine, and 2-methylpyridine suddenly increase with decrease of temperature from 330 to 300 K.28 These features would be due to structural change. Conformation change forward compact structure enhances the reaction. Unlike ubiquitin and angiotensin I ions, for [Mi + zH]z+, conformation change forward compact structure only reduces the reaction. Therefore, many variables that suppress or promote proton transfer are intricately intertwined. Williams’ group has reported reaction rate constants of proton transfer from [Mi + zH]z+ and [Mr + zH]z+ to gaseous molecules by means of FT-ICR.61 Because PA of dipropylamine is almost equivalent to that of Dmpy,74 the present results of the rate constants with Dmpy can be compared with their results for the reaction with dipropylamine. For [Mr + 10H]10+, the present values were almost the same as their results. For [Mi + 8H]8+, [Mi + 9H]9+, and [Mr + 9H]9+, the present values were larger than their results. In the present measurements, there were a number of thermal collisions with He. On the other hand, they have measured them in ultrahigh vacuum of ICR cell, in pressure range of 10−7 Pa. They have determined PA and GB of protein ions from the measurements. Their GB values of [Mi + zH]z+ increase more suddenly with increase of charge state than those of [Mr + zH]z+.61 However, the present results show that the rate constants depend on temperature, and PA and GB also seemed to be dependent on temperature. The present experimental results were roughly consistent with IMS works. However, it was not perfectly consistent. This is because IMS works relate to whole structure,3−5,20−23,57−60,62 whereas the rate constants of proton transfer would relate to local structure, local environment of protons bound to basic residues and surrounded by hydrophilic sites. It is expected to give another kind of aspect to characterize the protein ions. IMS works have reported that there are plural conformers. In the present works, specific charge states were selected, but specific conformers were not selected, and averaged features including over all conformers at a specific charge state were measured. IMS works have also reported that protein conformations are dependent on solution composition.5,20,21 In the present study, lysozyme was dissolved in methanol− water mixture including acetic acid, and its initial gas-phase conformations are supposed to be partially unfolded. However, the conformations are thermalized by collision with He during trap in the gas cell. Based on this assumption, observed branching fractions are represented. Details are described in the next section. Representation of Observed Branching Fractions. Experimental features for observed branching fractions were represented by calculated reaction rate constants with a scheme of sequential process. Details of the scheme have been described elsewhere.29 Proton transfer proceeds as a sequential reaction in the following k1

k2

k3

k4

k5

Pz → Pz − 1 → Pz − 2 → Pz − 3 → Pz − 4 → . . .

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Initial condition is described in the following: for t = 0, [Pz] = [Pz]0 = 1, and [Pz−1] = [Pz−2] = [Pz−3] = [Pz−4] = 0. Parent ions are transmitted into the gas cell continuously, whereas parent and product ions are extracted out as pulses. Therefore, branching fractions are averaged features of various retention times. By means of the scheme described above, the observed branching fractions were represented by the rate constant of each ion, on the assumption of sequential process. These results are presented in Figures 2b and 4b. Error propagation was estimated with errors of the rate constants. Comparing Figure 2b with Figure 2a, similar features were obtained. Shapes of curved lines were similar. The representation was good enough. From these results, it was confirmed that product ions were generated by way of sequential reactions. The rate constants are related with conformation of ions. Product ions, [Mi + z′H]z′+ (z′ = 7−10), were generated in the gas cell by proton transfer, whereas a parent ion, [Mi + 11H]11+, was generated with ESI and was transmitted into the gas cell. Figure 2a,b shows that line shapes for [Mi + z′H]z′+ are similar. It means that the rate constants of [Mi + z′H]z′+ are similar to that of [Mi + zH]z+. This is because line shapes in Figure 2b are based on measured rate constants of [Mi + zH]z+. Namely, line shapes of [Mi + z′H]z′+ and [Mi + (z′ − 1)H](z′−1)+ in Figure 2b depend on measured rate constants of [Mi + zH]z+ selected as parent ions. Therefore, conformations of [Mi + z′H]z′+, which are produced by sequential process, are similar to those of [Mi + zH]z+, which are injected into the gas cell as parent ions. Similar results were obtained in the reaction of [Mi + 9H]9+ to those for [Mi + 11H]11+. The shapes of curved lines of each charge state are similar. The observed and represented branching fractions of the parent ion, [Mi + 9H]9+, and product ions, [Mi + z′H]z′+ (z′ = 6−8), are plotted as a function of temperature in the gas cell in Figure S-3 in the Supporting Information. In the reactions of [Mi + 8H]8+ and [M i + 10H] 10+ , similar features were obtained. The representation was fairly good enough. Therefore, in the reactions of [Mi + 8H]8+, [Mi + 9H]9+, [Mi + 10H]10+, and [Mi + 11H]11+, the product ions were generated by way of sequential reactions. In these reactions, the conformations of [Mi + z′H]z′+ were similar to those of [Mi + zH]z+. Similarly, in the reaction of ubiquitin ions with 1-butylamine, the representation has been fairly good, except for the 7+ charge state.29 For the 7+ charge state of the ubiquitin ion, thermalized conformation of product ion would be different from that of parent ion at low temperature range. Comparing Figure 4b with Figure 4a, almost similar features were obtained. Shapes of curved lines of 12+, 13+, and 14+ charge states were similar. From these results, in the reactions of [Mr + 14H]14+, product ions were generated by way of

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where Pz is parent ion as a reactant and Pz−1, Pz−2, Pz−3, Pz−4, ... are product ions. Reaction rate constants, k1, k2, k3, k4, ..., include density of the target molecule, [M] (molecule cm−3). They are k1 = k′1[M], k2 = k′2[M], k3 = k′3[M], and k4 = k′4[M]. [M] is assumed to be in significant excess of the [(M + zH)z+]. Assuming that Pz−4 is not reactive and k5 is equal to 0, a set of simultaneous differential equation is given as 9658



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product ions were represented successfully by calculated reaction rate constants with a scheme of sequential process. On the other hand, for [Mr + zH]z+, at low temperature range, the observed branching fractions for product ions of lower charge states were not represented. These features are closely related to the protein conformations stabilized by disulfide bonds.

sequential reactions. However, the shapes of curved lines of lower charge states, 10+ and 11+, were not similar. In particular, the line shape of [Mr + 10′H]10′+ in Figure 4a was far from that in Figure 4b at lower temperature range. The branching fraction of [Mr + 10′H]10′+ in Figure 4a increased gradually with decrease of temperature, whereas that in Figure 4b increased drastically. The line shape of [Mr + 10′H]10′+ relates to the rate constant of [Mr + 11′H]11′+. It means that the rate constant of [Mr + 11′H]11′+ was not similar to that of [Mr + 11H]11+. Therefore, conformation of [Mr + 11′H]11′+ would be different from that of [Mr + 11H]11+ at low temperature range. In other words, thermalization to equilibrium conformation for [Mr + 11′H]11+′ would not finish in the gas cell. In this point of view, conformation of [Mr + 11′H]11+′ has a “memory” to remember its origin of production, carrier and history. In this experiment, maximum retention time in the gas cell was 100 ms. We suppose that it is long enough for thermalization, except for [Mr + 11′H]11+′ at low temperature range. The time required to achieve an equilibrated population of gas-phase [Mr + 11′H]11+′ structures at low temperature appears to exceed 100 ms. Similar results were obtained in the reaction of [Mr + 12H]12+. The observed and represented branching fractions of the parent ion, [Mr + 12H]12+, and product ions, [Mr + z′H]z′+ (z′ = 8−11), are plotted as a function of temperature in the gas cell in Figure S-4 in the Supporting Information. Comparing observed and represented branching fractions, the shapes of curved lines of the parent ion and product ions of higher charge states were similar, whereas those of lower charge states were not similar at the lower temperature range. Similar results were obtained in the reactions of [Mr + 10H]10+, [Mr + 11H]11+, and [Mr + 13H]13+. These results are not shown here. Consequently, thermalization of [Mr + z′H]z′+ is much slower than that of [Mi + z′H]z′+. Unfolding and refolding properties of lysozyme in the solution phase have been investigated by a wide range of complementary biophysical techniques.44−46 Refolding time of disulfide-reduced lysozyme is much slower than that of disulfide-intact lysozyme. In the gas phase as well as in the solution phase, amputation of disulfide bonds reduces thermal stability of the protein conformation. The relationship between disulfide bonds and conformation stability is a common feature. Namely, the disulfide bonds stabilize folded conformation of lysozyme in the gas phase as well as in the solution phase.

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ASSOCIATED CONTENT

S Supporting Information *

Details of experimental apparatus used in the present study, and details of electrospraying disulfide-intact and disulfide-reduced lysozyme solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was partly supported by the Grant-in-Aid for Scientific Research in the priority area “Molecular Science for Supra Functional Systems” (no. 0920050025) from MEXT. The authors thank Prof. T. Kondow and Prof. K. Fuke for their kindhearted help with constructing the apparatus used in the present study. The authors are grateful to graduate students in our group, Mr. T. Matsuda, Mr. T. Ito, Mr. Y. Oguchi, and Mr. Y. Yokoyama, for making a number of ion optics and electric circuits which compose the apparatus. The authors also thank Prof. M. Takayama, and Prof. K. Honma for helpful discussions.

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CONCLUSIONS Gas-phase proton-transfer reactions of disulfide-intact and -reduced lysozyme ions, which were produced with electrospray ionization, to 2,6-dimethylpyridine were studied using tandem mass spectrometry. By changing temperature of the gas cell, the temperature dependence of reaction rate constants and observed branching fractions for proton transfer was measured. Absolute reaction rate constants for proton transfer for the protein ions of specific charge states were determined. Dramatic changes were obtained for reaction rate constants and distribution of product ions. The rate constants grew larger with increase of charge state. For disulfide-intact ions, [Mi + zH]z+, they increased more rapidly with increase of charge states than those for disulfide-reduced ions, [Mr + zH]z+. Exceptionally, the reaction rate constant of [Mi + 7H]7+ was much larger than that of [Mi + 8H]8+ over all temperature range. With change of temperature, the rate constants for [Mi + zH]z+ changed more drastically than those for [Mr + zH]z+. For [Mi + zH]z+, observed branching fractions for parent and 9659



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Conformations of Disulfide-Intact and-Reduced Lysozyme Ions Probed

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