pH Dependence of the Number of Discrete Conformers of Carbonic Anhydrase 2, as Evaluated from Collision Cross-Section Using Ion Mobility Coupled with Electrospray Ionization

Ion mobility experiments coupled with electrospray ionization (ESI) were conducted to evaluate the folding states of bovine carbonic anhydrase 2 (CA2) under three different pH conditions. Collision cross-section (CCS) of the CA2 ions generated by ESI revealed the presence of six discrete conformers in the gas phase under the conditions employed in this study. The CCS of the most extended conformer was three times larger than that of the most compact one. The charge state distribution of the CA2 ions was indicative of three conformers being present. Although there was consistency in conformer assignment conducted by CCS and charge state distribution, the CCS measurement was shown to be more effective because the information obtained provided more detailed knowledge of the conformation of the protein.


INTRODUCTION
One of the commonly accepted methods of analyzing protein folding states by mass spectrometry (MS) is to monitor the shi in charge state distribution of multiplyprotonated protein molecules, [M+nH] n+ , generated by electrospray ionization (ESI), [1][2][3][4][5][6][7] because ESI can directly transfer those ions from the solution to the gas phase. Although the sample solution passes through an electrospray needle with a high electric eld, it is thought that analytes remain in solution until the nal ionization step. 8) If a protein is in a folded conformation, ESI produces gaseous ions with a relatively small charge number, because compactly folded polypeptide chains only allow protonation at the basic side chains that are exposed at the protein surface during the ionization process. In contrast, for an unfolded protein, a signi cantly large number of protons on the polypeptide chain are accessible, and the ions are shi ed to a lower m/z region. [2][3][4] A multimodal charge state distribution suggests the coexistence of several conformers. [1][2][3][4][5] On the other hand, ion mobility spectrometry (IMS) coupled with ESI is now recognized as a powerful method for analyzing protein conformations in the gas phase, because IMS can separate ions having the same m/z values but with di erent shapes or sizes. e dri time measured for an ion can be converted into a corresponding collision crosssection (CCS), and the CCS provides information related to the conformation of the protein. [9][10][11][12][13][14][15][16] We have evaluated the folding states of bovine carbonic anhydrase (CA2) in the gas phase. CA2 is a metalloprotein that catalyzes the reversible hydration of CO 2 , in other words, CO 2 +H 2 O↔HCO 3 − +H + . [17][18][19] CA2 consists of 259 amino acids, in which a Zn 2+ ion is bound to the active center by three histidine-imidazoles and a H 2 O, 17) and is involved in various physiological functions, such as respiration, pH regulation, CO 2 and HCO 3 − transport, and bio mineralization. 20,21) We previously reported on the e ects of solution pH on the charge state distribution of CA2 ions produced by ESI and demonstrated that monitoring the m/z change caused by the removal of the Zn 2+ it was possible to observe the conversion from holo-CA2 to the apo-form. 22) From the product ion spectra obtained under several different solvent conditions, we showed that the folding states of the C-terminal region of the protein were in uenced by the solvent. 23,24) However, the relationship between the folding states of CA2 ions and their molecular size have not yet been examined.
In the present study, we used a mass spectrometer equipped with a traveling wave ion mobility system to measure the mobility of multiply-charged CA2 molecules generated by ESI at three di erent pH conditions. e pH dependence of the number of conformers and the CCS values observed for each charge state was evaluated, and the folding states of the CA2 ions were elucidated from the CCS obtained at di erent pH conditions. e results were compared to the folding states evaluated from the charge state distribution.

Materials
CA2 from bovine erythrocytes was purchased from Sigma (St. Louis, MO). Equine myoglobin and equine cytochrome c, which were used as calibrants for CCS measurements, were obtained from Sigma. All other reagents, such as acetic acid, ammonium acetate, and methanol were of the highest grade available and obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Puri ed water was prepared by Milli-Q Advantage A10 (Merck Millipore, Billerica, MA).

Sample preparation
An approximately 100 pmol/µL solution of CA2 was prepared by dissolving the protein in puri ed water. To dilute the CA2 solution for MS, the following solvents were prepared: (1) 0.1% formic acid (pH 2.6), (2) 0.3% acetic acid (pH 3.8), and (3) 20 mM ammonium acetate solution (pH 6.5). e pH of these solutions was adjusted with aqueous ammonia. e analyte CA2 concentration prepared to acquire ESI mass spectra was approximately 10 pmol/µL. e CA2 solutions were maintained at ambient temperature for at least 15 min to complete the conformational shi and the Fig. 1. ESI mass spectra of CA2 measured at three di erent pH conditions. A CA2 concentration of approximately 10 pmol/µL was used for the measurements. CA2 was dissolved in (a) 0.1% formic acid (pH 2.6), (b) 0.3% acetic acid, adjusted to pH 3.8, (c) 20 mM ammonium acetate (pH 6.5). Bi-and tri-modal charge state distributions were observed at pH 2.6 and 3.8, respectively. e holo-CA2 ions were dominantly observed in pH 6.5. Asterisks indicate ions that are both holo-and apo-CA2. ree ensembles A-C were assigned from the charge state distribution. samples were then infused into a mass spectrometer.

Mass spectrometry
Mass spectra were obtained with a SYNAPT G2-Si HDMS quadrupole IMS orthogonal acceleration time-ofight mass spectrometer equipped with an electrospray ion source and a MassLynx data processor (Waters Corp., Milford, MA). e mass spectrometer was set to detect positive ions. e following data acquisition parameters were employed: electrocapillary voltage of 3.0 kV, sample cone voltage of 30 V, source temperature of 100°C, and desolvation temperature of 200°C. To measure ion mobility, nitrogen was used as a bu er gas, and IMS cell pressure was maintained at 3.1 mbar. e IMS wave velocity was 500 m/s and the wave pulse height was 35 V. e ow rate at which sample solutions were directly infused was 5.0 µL/min. e obtained dri times were converted into CCS values using the procedure outlined by Robinson et al. 9,25) Equine myoglobin and equine cytochrome c, each of which were dissolved in 49% (v/v) methanol containing 2% (v/v) acetic acid, were used as calibrants and the calibration data were obtained using the same IMS parameters as those for CA2. Myoglobin ions of 15+ to 24+ and cytochrome c ions of 13+ to 19+ were used for the calibration plot. e entire range of CCS was obtained from the formula derived from the calibration plot. e CCS for CA2 was calculated from the published X-ray structure 17) (RCSB PDB: 1V9E) using the MOBCAL so ware. 26,27) To estimate the number of independent conformers in ESI mass spectra, chemometric processing was conducted using the Origin 8.5 so ware (Origin Corp., Northampton, MA) as described previously. 28)

ESI mass spectra of CA2 at widely varying pH conditions
ESI mass spectra of CA2 obtained at three di erent pH conditions are shown in Fig. 1. At pH 2.6, two local maxima at 20+ (m/z 1452.2) and 34+ (m/z 854.6) and a local minimum at 25+ (m/z 1162.0) were observed in a bimodal charge state distribution of [M+nH] n+ corresponding to apo-CA2 (Fig. 1a). In the spectrum obtained at pH 3.8, ions corresponding to both apo-CA2 and holo-CA2 were detected. e multiply-charged holo-ions, [M+nH+Zn] n+ , were observed from 10+ to 20+ and the apo-ions [M+nH] n+ were observed at all the charge numbers except 10+. In the spectrum, a trimodal charge state distribution was observed with local minima at 14+ (m/z 2074.0 for the apo-ion and m/z 2078.6 for the holo-ion) and 28+ (m/z 1037.5 for the apo-ion), as shown in Fig. 1b. e distributions of the apoand holo-ions were not correlated with the trimodal charge state distribution. At pH 6.5, holo-CA2 ions from 10+ to 12+ were observed with the maximum at 11+ (m/z 2645.2, Fig. 1c).
We also conducted a chemometric analysis to determine the number of independent conformers in the multimodal charge state distribution of ESI mass spectra (Fig. 1). e results obtained suggested that CA2 ions were composed of three components for the apo-ions (A-C) and two components for the holo-ions (B and C) under the conditions used in this study (Fig. 2).
is indicates that apo-and holo-CA2 consist of three and two conformers, respectively. e enzymatic activity of CA2 is known to be proportional to the binding rate of Zn 2+ , and the enzyme has both hydration and dehydration activities at pH 6.5. 29) e CA2 ions observed at pH 6.5 were holo-CA2 ions and belonged to component C. erefore, component C re ects the folded conformation of CA2, while the ions comprising component A are all in the apo-form. Indicating that the component likely consists of inactive extended conformers. On the other hand, component B was observed at pH 2.6 and pH 3.8 but not at pH 6.5, indicating that ions belonging to this component do not represent physiologically active conformers. At pH 3.8, component B contains some holo-CA2 ions e results of deconvolution of charge state distribution of CA2 ions in ESI mass spectra obtained at three di erent pH conditions. e raw data are shown in Fig. 1. Presence of three components was indicated.
with lower charge numbers. It can be concluded that component B consists of apo-and holo-ions, suggesting that the component might be in a transition state to component A which is dominantly observed at pH 2.6.

pH dependence of the number of discrete conformers observed in IMS experiments
Ion mobility experiments were performed to obtain information concerning the gas-phase conformation of the  CA2 ions at pH 2.6, 3.8, and 6.5. e dri grams obtained are shown in Fig. 3. e singlet peak observed in higher charge numbers 29+ to 47+ at pH 2.6 suggests that the ions consist of only one conformer. e peak shape became broader as the charge number decreased, and doublet peaks clearly appeared from 25+ (Fig. 3a). At pH 3.8 for apo-ions ( Fig. 3b-1), a singlet peak in the higher charge number ions and doublet peaks from 25+ were observed. e presence of more than two peaks was assumed, since the charge number decreased, suggesting the presence of several conformers. For holo-ions (Fig. 3b-2), the number of the peaks observed in each charge number ion was similar with that of the apo-ions, but the peak shape tended to become narrower than that of apo-ions in the higher charge state. is may be due to a stabilizing e ect of Zn 2+ on the conformation of the molecule. At pH 6.5 (Fig. 3c), doublet peaks were obtained at 12+ charge state, which were shi ed to a singlet peak at 10+. ese dri grams were similar to those of the holo-ion at pH 3.8. Comparing the dri grams obtained at three different pH conditions, the dri time and peak shape of ions with the same charge number were relatively similar to each other. is suggests that the charge number of the ions is a factor in determining the size of CA2 ions. e pH dependence of the CCS on the conformers observed by IMS e peak top CCS of the dri grams was obtained for the three pH conditions and the CCS was plotted against the charge number of the ions, as shown in Fig. 4. From the plot, the peak tops could be divided into six groups, indicating the presence of six conformers (I-VI). At pH 2.6, conformers I and II were predominant, while conformers V and VI were predominant at pH 6.5. e remaining conformers III and IV were mainly observed at pH 3.8.
e CCS values for the peaks observed on the dri grams at each charge number were calculated, and the intensity of the ions was plotted against the obtained CCS (dotted line in Fig. 5). e sum total of the intensity of each plot is shown as a solid line in Fig. 5. In the case of the sum total plot at pH 2.6, two conformers I and II corresponding to apo-CA2 were found with CCS values that ranged from 50 to 120 nm 2 (Fig. 5a), and the top of the peak of conformer I was 87 nm 2 . Both the range of CCS values and peak widths of these conformers were much wider than the other conformers, indicative of the extended shape of conformers I and II. In the case of pH 3.8 apo-ions, the CCS distribution showed a wide range from 25 to 105 nm 2 and more than 50 nm 2 ions were abundant. Conformers I, II, and III were contained in this abundant region. In the region less than 50 nm 2 , three conformers corresponding to IV, V, and VI were considered to be present but conformers V and VI were not observed for a speci c peak ( Fig. 5b-1). In the case of pH 3.8 holo-ions, the CCS distribution showed a wide range from 25 to 65 nm 2 and the presence of four conformers, III to VI was evident ( Fig. 5b-2). A clear valley at 40 nm 2 between conformers IV and V was observed.
is indicates that these conformers may be separated by an energy barrier. e CCS distribution for conformers V and VI was similar to that at pH 6.5 (Fig. 5c), indicating that the folding states of conformers V and VI at pH 3.8 holo are similar to those at pH 6.5. At pH 6.5 (Fig. 5c), ions corresponding to holo-CA2 were revealed to be composed of two conformers, V and VI. e CCS for these two conformers was between 20 and 40 nm 2 , and the peak top for conformer VI was approximately 27 nm 2 . is CCS value is consistent with the value taken from X-ray crystal structure calculated via the MOBCAL trajectory method, indicating 24 nm 2 .
is suggests that the folding state of conformer VI may be similar with that of the crystal structure. Holo-ions observed at pH 3.8 and 6.5 were distributed in a smaller CCS region than that for apo-ions. is suggests that the coordination of Zn 2+ may contribute to maintaining the compact shape of the ions.

CONCLUSION
Ion mobility experiments of multiply-charged CA2 ions generated by ESI at three di erent pH conditions indicated that the gas phase CA2 ions have six discrete conformers (I-VI) with CCS ranging from 20 to 120 nm 2 under the condition employed in this study. e CCS of peak top of the most compact component VI was 27 nm 2 , whereas that of most extended component I was 87 nm 2 . e CCS of the most compact conformer was consistent with that of the X-ray crystal structure. e conformation of the CA2 ions was analyzed by evaluating the charge state distribution and the results suggest that three components in the apoions and two components in the holo-ions are present (A-C, Fig. 2), whereas mobility measurements showed the presence of six conformers (I-VI). e mobility and CCS values reported here suggest that component A included conformer I, component B included conformers II, III, and IV, and component C included conformers V and VI (Figs. 2 and 5). Although there was consistency between the methods, mobility measurements were shown to be more e ective because the information obtained gave more detailed information regarding the conformation of the protein.