Mechanism of Protein Molecule Isolation by IR Laser Ablation of

Article pubs.acs.org/JPCA

Mechanism of Protein Molecule Isolation by IR Laser Ablation of Droplet Beam Kensuke Komatsu, Takuya Nirasawa, Mariko Hoshino-Nagasaka, and Jun-ya Kohno* Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan ABSTRACT: Gas-phase isolation of bovine serum albumin (BSA) from aqueous solutions is performed by IR laser ablation of a droplet beam. Multiply charged BSA ions (positive and negative) were produced by the IR laser irradiation onto a droplet beam of aqueous BSA solutions with various pH values prepared by addition of hydrochloric acid or sodium hydroxide to the solution. The isolation mechanism was discussed based on the charge state of the isolated BSA ions. A nanodroplet model explains the gas-phase charge distribution of the BSA ions. This study provides a fundamental basis for further studies of a wide variety of biomolecules in the gas phase isolated directly from solution.

1. INTRODUCTION Biological molecules function in a living body in cooperation with adjacent molecules including the water solvent. For example, some protein molecules adsorb molecules to carry or store them in an appropriate position of the living body. Such particular function results from folded structures of the protein molecules, which are determined by interactions with solvent water molecules.1 Therefore, it is important to investigate interactions of proteins and water molecules. The molecular function of the water molecules can be scrutinized by observing properties of protein molecule as a function of the hydration number. In such studies, it is important to isolate the hydrated protein molecules in the gas phase. Gas-phase isolation and subsequent analysis of protein molecules have been achieved by various methods, such as electrospray ionization2−5 and matrix-assisted laser desorption/ ionization.6,7 Among them, we use IR laser ablation of droplets in a vacuum: A droplet of an aqueous solution in vacuum is irradiated with an IR laser resonant with the OH stretching mode of liquid water. The molecules in the solution are ejected from solution into the gas phase without suffering significant degradation. This method was explored by Brutschy and coworkers8−13 followed by Abel and co-workers14−17 and ourselves.18−28 Recently, we have developed a droplet-beam laser ablation mass spectrometry (DB-LAMS) apparatus which enables us to reduce the sample consumption by use of a pulsed injection of the solutions to vacuum. We confirmed that the droplet exists in the liquid phase in vacuum26 and investigated a UV-laser-induced proton-transfer reaction of lysozyme molecules in the gas phase.27 Further instrumental development is in progress, where the isolated ions are trapped for long times in a Paul trap to apply gas-phase spectroscopies.28 Elucidation of the mechanism for IR laser ablation of the droplets is necessary to discuss the gas-phase molecular properties in relation to the molecular functions in the solutions. Abel and co-workers have proposed a nanodroplet model for the isolation mechanism.16 Their model assumes that © XXXX American Chemical Society

liquid IR laser ablation produces nanosized liquid droplets, in which the solute ions of interest is included, and the nanodroplets result in product ions by evaporation of water molecules. In our previous paper, we revealed that the nanodroplet model can describe the isolation mechanism of lysozyme ions produced by IR laser ablation of the droplet beam.29 We discussed the difference in the charge distributions of lysozyme in the solution from those in the gas phase on the assumption that the ions randomly distribute to the nanodroplets and hence show a Poisson distribution. Our work has been followed by Abel and co-workers, who describe the gasphase ion yield from a NaCl aqueous solution with the model.17 For more general discussion, however, our work on lysozyme29 is insufficient because lysozyme has extremely high isoelectric point (pI), >11,1 and hence is protonated in the solution. Our previous work is therefore limited for positive ions isolated into the gas phase. In the present paper, we employ bovine serum albumin (BSA) as a sample in order to determine whether the nanodroplet model works for other protein molecules. The BSA molecule is selected because BSA has moderate pI (4.9), and its structure is already solved. Therefore, we can discuss the isolation mechanisms of both positive and negative ions from solutions with wide pH range by use of BSA. We propose a method to calculate the charge distribution of any protein molecule based on its molecular structure and show general applicability of the nanodroplet model. The comparison of the difference in the charge distributions in the solution phase from those in the gas phase can be described by the nanodroplet model semiquantitatively.

2. EXPERIMENTAL SECTION A detailed description of DB-LAMS has been given previously.26 We briefly describe the apparatus and the Received: November 6, 2015 Revised: February 19, 2016

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DOI: 10.1021/acs.jpca.5b10873 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry A

protonated and deprotonated groups and can be associated with ions such as Na+ and Cl− as well as water molecules. However, the mass resolution is insufficient to determine the number of these species associating with the ions, so the peaks unify to a single peak. Ions assignable to H+·(H2O)k and Na+· (H2O)k (for positive ions) and OH−·(H2O)k and Cl−·(H2O)k (for negative ions) appear in the low m/z region (not shown in Figure 1) in the mass spectra at higher intensities. As seen in Figure 1, the intensity of [BSA]n+ significantly increases when HCl is added to the aqueous BSA solution and decreases when NaOH is added, whereas that of [BSA]n− decreases when HCl is added and increases when NaOH is added to the solution. Figure 2 shows the charge distributions of BSA in aqueous solutions of (a) 20 μM BSA and 100 μM HCl, (b) 20 μM BSA,

experimental procedures employed in the present study. The apparatus consists of a piezo-driven liquid droplet nozzle and a reflectron time-of-flight mass spectrometer (TOF-MS), which is housed in a three-stage differentially pumped vacuum chamber. A droplet (∼70 μm in diameter) of a sample solution was injected into air from the nozzle. We employed aqueous solutions of 20 μM bovine serum albumin (BSA) or lysozyme (Lys) mixed with x μM hydrochloric acid (HCl) or sodium hydroxide (NaOH) (x = 0−1000) and y mM NaCl (y = 0−10). Commercially available BSA (Wako Chemicals), Lys (Seikagaku Biobusiness), hydrochloric acid, NaOH, and NaCl were used without further purification. Deionized and distilled water was used as the solvent. Sample solution pH values were obtained using a pH meter (Horiba Scientific, B-71X). Gas flow from the inlet aperture to the first vacuum chamber carries the droplet through the second chamber to the third, where the droplet is admitted into the acceleration region of the TOF-MS. Multiply charged protein ions are produced from the droplet by IR laser irradiation (3586 cm−1, ∼5.5 mJ pulse−1) resonant to the OH vibrational mode of liquid water. Product ions were accelerated by a pulsed electric field (2.1 kV) and were analyzed by the TOF-MS after a 2 μs delay time. The delay time was set to obtain higher mass resolution. The apparatus employed a Daly detector to minimize the deterioration of the detection efficiency for the high m/z region, such that we successfully observed singly charged BSA ions (m/z ∼ 66 000) in the present experiment. The apparatus has an intrinsic mass resolution of 400.26 However, the BSA peak consists of so many mass-unresolved species (see section 3) that the apparent mass resolution seems to be less than the intrinsic one.

3. RESULTS Figure 1 shows mass spectra of positive and negative ions obtained from aqueous solutions of (a) 20 μM BSA and 100 Figure 2. Charge distributions of BSA in aqueous solutions of (a) 20 μM BSA and 100 μM HCl, (b) 20 μM BSA, and (c) 20 μM BSA and 100 μM NaOH and those species isolated from the solutions into the gas phase. The red, blue, and green traces represent the intensities of positive ions in the gas phase, negative ions in the gas phase, and ion abundance in the solution, respectively.

and (c) 20 μM BSA and 100 μM NaOH and species isolated from these solutions into the gas phase. The charge distributions for the solutions are calculated results. We describe the calculation method of the solution charge distribution in section 4.1. The gas-phase charge distributions are obtained from the experimental results shown in Figure 1. The charge distribution in the solutions shifts toward the positive or negative directions by the addition of HCl or NaOH to the solution, presumably because of protonation or deprotonation of the side groups of the BSA molecule. However, the number of charges on the BSA molecules (positive or negative) in the gas phase is significantly smaller than that in the solutions. Figure 3 shows the total intensity of [BSA]n+ (1 ≤ n ≤ 6) and [BSA]n− (1 ≤ n ≤ 4) as a function of solution pH. The intensities of the positive and the negative ions are normalized at their maxima because the detection efficiency is different for the positive and the negative ions. The total intensity of [BSA]n+ has a large value at pH ≤ 4, steeply decreases at pH 4.5−5.1, and levels off as the solution pH is further increased. However, the total intensity of [BSA]n− has a small value at pH

Figure 1. Mass spectra of positive (left) and negative (right) ions obtained from the droplet beam of aqueous solutions of (a) 20 μM BSA and 100 μM HCl, (b) 20 μM BSA, and (c) 20 μM BSA and 100 μM NaOH under IR laser irradiation at 3986 cm−1 and 5.5 mJ pulse−1.

μM HCl, (b) 20 μM BSA, and (c) 20 μM BSA and 100 μM NaOH under IR laser irradiation on the droplet beam. The IR laser power is set to 5.5 mJ pulse−1. Peaks in the mass spectrum are assigned to [BSA]n+ (1 ≤ n ≤ 6) and [BSA]n− (1 ≤ n ≤ 4) for the positive and the negative ions, respectively. Ions assignable to [BSA2n+] (n = 3, 5) are observed with small intensities. The ions, [BSA]n+ and [BSA]n−, may have B


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processes. The ion intensity may saturate at the high IR laser intensity region. The positive ions produced from 20 μM BSA + 500 μM HCl (panel a) look to level off above 6 mJ/pulse. However, analysis of the data at 3.5−6 mJ/pulse gives the k value of 1.1. This value still indicates that the ionization is a single-photon process.

4. DISCUSSION 4.1. Charge Distribution of BSA in Aqueous Solution. We discuss the isolation mechanisms of ions from solution into the gas phase by considering the charge distributions of the BSA ions. First, the BSA charge distribution in solution is calculated as a basis of the discussion. In the previous paper, we calculated the charge distribution of another protein molecule, lysozyme, in solution,29 which was obtained on the assumption that each of the lysozyme side chains are in independent equilibria: We calculate the molar ratio of the protonated/ deprotonated forms of all the electrolytic side chains of the constituent amino acids from their pKa values and the solution pH. Here, the pKa values are equilibrium constants of the reaction

Figure 3. Total intensity of [BSA]n+ (1 ≤ n ≤ 6) (red) and [BSA]n− (1 ≤ n ≤ 4) (blue) as a function of the solution pH. Lines represent ion intensities with a model calculation.

≤ 4.5, a steep increase at pH 4.7−5.3, and levels off with further increases in the solution pH. The solid curves in Figure 3 show ion intensity values calculated using a model described in section 4.2. Figure 4 shows the total ion intensity of BSA ions isolated from the droplet beam as a function of the incident IR laser

HA ⇋ A− + H+

(2) 29

where A shows the constituent amino acid. The number of charges on the lysozyme molecule is then obtained from the molar ratios. The calculation gives a reliable result because (1) all the pKa values of the 32 electrolytic side chains are reported and (2) the result reproduces the isoelectric point (pI, ∼11 for lysozyme) reasonably well. In comparison to the previous study on lysozyme, however, we have to introduce another way to calculate the charge distribution of BSA in solution because pKa values of the individual BSA side chains are unknown. Initially we assume that all the electrolytic amino acid BSA side chains have representative reported pKa values.1 However, this calculation gives the isoelectric point (pI) of BSA as 5.4, which differs with the experimental value (4.9).1 This discrepancy comes from the faulty assumption that all similar amino acid side chains have the same pKa values because the pKa values of the side chains depend on their environments (hydrophilic or hydrophobic). The BSA molecule has a folded structure in solution. Therefore, amino-acid residues located in the inner part of the BSA molecule are not in contact with solvent water molecules and no longer exhibit the same protonation/deprotonation equilibrium. To estimate the charge distribution of the BSA molecules, we introduce a parameter, the accessible surface area (ASA) proposed by Lee and Richards.30 ASA is a parameter calculated for each amino acid component of a protein molecule based on its folded structure and represents the area with which solvent water molecules are in contact. Large ASA values indicate that the side chain has extensive contact with solvent water molecules and hence is likely to display its intrinsic pKa value, while low ASA values indicate that the side chain is located in the inner, solvent-sparse region of the BSA molecule which favors the noncharged form. ASA values are calculated for each amino acid side chain from the X-ray diffraction structure of the protein molecule. To calculate the ASA of BSA, we used the PDB database structure 4F5S and the StrucTools software.31 A probe radius of 1.4 Å was used. We set a threshold ASA value, tASA, and calculate pIs on the assumption that amino acids with larger ASA values than tASA are in equilibria and that those with lower ASA values exist in

Figure 4. Total ion intensity of BSA ions isolated from the droplet beam of (a) 20 μM BSA and 500 μM HCl and (b) 20 μM BSA and 200 μM NaOH as a function of the incident IR laser power. Blue and red plots represent the positive and the negative ions, respectively.

power. Panels a and b depict results obtained from solutions of 20 μM BSA and 500 μM HCl (pH 4.16) and 20 μM BSA and 200 μM NaOH (pH 5.96), respectively. These solutions conditions were chosen from the plateau region of the pH dependence of the ion intensity shown in Figure 3. The power dependences are linear in the double-logarithmic plot, which shows that the ion intensities are represented as (1) I = AP k where I and P are the gas-phase ion intensity and the IR-laser power, respectively, and A and k are parameters. In this analysis the parameter k represents the number of IR photons required for BSA ion formation. By fitting the experimental values to eq 1, we obtained k values of 0.8 ± 0.2 and 0.5 ± 0.1 for positive ions from the solutions of 20 μM BSA + 500 μM HCl and 20 μM BSA + 200 μM NaOH, respectively, and values of 1.0 ± 0.3 and 0.9 ± 0.1 for negative ions from the same solutions, respectively. The obtained values indicate that ion formation is a single-photon process and does not include IR multiphoton C


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according to a nanodroplet model, originally proposed by Abel and co-workers.16 Briefly, the droplet which absorbs the IR laser disintegrates into gas and/or nanodroplets via supercritical gas expansion, explosive thermal decomposition, and shockwave dispersion. The disintegration process includes an inhomogeneous heat-up of the liquid, which results in nonthermal desorption of molecules, clusters, and nanodroplets into the gas phase. The water molecules in the nanodroplet evaporate and result as product ions in the gas phase. Hence, ions are likely to be produced as an aggregate of the constituent ions in the nanodroplet, which indicates that a part of the multiple charges on the BSA ions is neutralized by counterions. This neutralization causes a decrease in the number of charges on the BSA molecules as a result of isolation in the gas phase, which is observed in the present experiment (see Figure 2). Because the nanodroplet model does not include multiphoton absorption of the IR laser, the ion abundance in the gas phase is likely to depend linearly on the IR laser power. This is observed experimentally (Figure 3), which supports our consideration on this model. Semiquantitative calculation of the BSA ion abundance in the gas phase is performed on the basis of the nanodroplet model. We follow the basic idea proposed in our previous study29 for the calculation but extended to the calculation of both positive and negative ions. In brief, nanodroplets produced by the IR laser ablation lose their solvent water molecules by evaporation and result into product ions. Ions are assumed to distribute randomly in the solution, and the number of the ions in the nanodroplet shows Poisson distribution statistics.31 We calculate, for an example of the ions isolated from a BSA + HCl solution, the abundance of a nanodroplet including a BSA2+ ion and a Cl− ion according to the statistics and count it as a part of the gas-phase abundance of BSA1+. We account for ions with opposite charged ions to the BSA ion for the model calculation because the ions with the same sign are likely to be repelled from the multiply charged BSA ions. Then, summing up all the possible n and m combinations for BSAn+·mCl− (BSAn−·mNa+ for negative ions), we obtain charge distributions of the BSA ions in the gas phase. The calculated results are shown as solid and dotted curves in Figure 3. The solid curves successfully reproduce the BSA ion intensity in the gas phase. The calculation, on the other hand, underestimates the intensities of BSAn+ or BSAn‑ for for solutions of pH < 4.2 or pH > 9.4, respectively (dotted curves in Figure 3), because these solutions are out of range of this model calculation: As described, the calculation ignores the ions having the same sign with the multiply charged BSA ions (H+, Na+ for BSAn+ and OH−, Cl− for BSAn‑) on the assumption that these ions repel each other by Coulomb repulsion. However, the amount of the counterions, Cl− or Na+, exceeds that of BSAn+ or BSAn− for solutions with pH < 4.2 or pH > 9.4, respectively. In such solutions, the ions with the same sign with the BSA ion are contained in the nanodroplet without suffering from the Coulomb repulsion. According to the nanodroplet model, the neutralization of BSA would be minimal when the sample solution is extremely dilute and free from the salts, and hence the charge distribution would be wider, which might be an evidence for the nanodroplet model. However, it is difficult to observe it experimentally because the ion intensity decreases with the decrease in the BSA concentration. 4.3. Salt Addition. As shown in section 4.2, the ions in the solution are isolated into the gas phase by the IR laser ablation.

neutral forms and are excluded from the calculation. We only take into account the amino acids on the surface of the folded BSA molecule. The other calculation techniques refer to our previous study.29 Figure 5 shows the calculated pIs as a function

Figure 5. Calculated pIs as a function of tASA.

of tASA. The calculated pIs are dependent on tASA. The result indicates that the calculated pI reproduces the experimental value, 4.9, at four tASA values, such as ∼40, 57, 65, and 73 Å2. Figure 6 shows the calculated and measured pH levels of the

Figure 6. Calculated and measured pH levels of the solutions employed. Orange, red, blue, and purple lines represent the calculated values with tASA = 40, 57, 65, and 73 Å2, respectively, and blue dots represent experimental values.

solutions employed in the present study. The calculated result differs from the experiment when tASA is set to 40. On the other hand, the calculations qualitatively reproduce the experiment when tASA is set to 57, 65, or 73, but it does not trace it completely. The disagreement originates from our assumption that the amino acid residues with larger ASA than tASA are in equilibrium and that the others do not dissociate. Here, our discussion proceeds semiquantitatively on this inevitable ambiguity. Among the tASA values taken in the calculation, we chose the results of tASA = 57 Å2 because they give the smallest square deviation from the experimentally measured pH values. This calculation includes 139 electrolytic groups among 204 total in the BSA molecule. The calculated charge distribution is shown as the green trace in Figure 2, which shows that (1) the charge distribution shifts toward positive (negative) values by adding HCl (NaOH) to the solution and (2) the distribution has a broad range (more than 10). The latter result is different from the charge distribution of the gas-phase ions isolated by IR laser ablation, which we describe in the following sections. 4.2. Gas-Phase Ion Abundance Calculated by Nanodroplet Model. Previously, we described the charge reduction of lysozyme ions by isolation into the gas phase quantitatively D


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in the gas phase from 20 μM BSA + 250 μM NaOH solution. This solution does not contain any positive ions in the solution (see Figure 2). The results indicate that a charge inversion takes place resulting from isolation to the gas phase from solution. The charge inversion is described by attachment of excess counterions to the BSA ions during IR laser ablation. Because the formation mechanism is considered to be the same for the positive and negative ions, we discuss below the positive-ion formation from the solution without containing any BSAn+ ions by abstracting protons from the BSA molecules with the addition of NaOH to the solution. Another solution without any BSAn+ ions is that with a large amount of NaOH in it. In the solution, the BSA molecules abstract their protons to become BSAm− ions. The BSAm− ion attracts ambient Na+ ions through a Coulombic interaction potential to become chargeneutralized. Therefore, most of the BSAm− ions are produced in the neutral form in the gas phase by IR laser ablation because the nanodroplet has the neutralized BSA ion in it. A small amount of positive and negative BSA ions are produced in the gas phase if the nanodroplet statistically includes excess or less amount of counterions in the nanodroplet. The above conjecture is supported by the results from adding NaCl to the solution. In the addition of NaCl to the solution that does not contain any BSAn+ ions, the probability that the neutral nanodroplet includes the excess Na+ ions increases with increase in the Na+ concentration. However, the number of charges in the nanodroplet is likely to decrease with further increases in the Na+ concentration because both Na+ and Cl− ions are likely to be in the nanodroplet. Hence, the ion intensity is expected to increase and then decrease with increasing NaCl concentration, which is clearly observed in the experiment (see Figure 8). The Na+ ions which charge the nanodroplet are likely to locate a region free from the electric field of the BSA ion (neutral region) in the nanodroplet. A simple nanodroplet calculation shows the size of the neutral region. The calculation is performed assuming that the excess Na+ ions randomly distribute at the neutral region and charge the nanodroplet to result in the charge-inversed BSA ions in the gas-phase, which can reproduce the experimental result (solid line in Figure 8). The calculation shows that the neutral region has a volume equivalent to a 7 nm diameter sphere, which is much smaller than the size of the nanodroplet (26 nm) and is an equivalent volume as considering the surface region of the nanodroplet and a depth of ∼0.17 nm. This result indicates that the charge inversion is interpreted according to an attachment of excess Na+ ions onto the surface of the nanodroplet.

The nanodroplet model semiquantitatively reproduces the dependence of the gas-phase ion abundance on the solution pH. We further confirm the nanodroplet model by addition of salt (NaCl) into the solution. Based on the nanodroplet model, the salt addition is likely to increase the gas-phase ion intensity at low salt concentrations because the number of the ions produced from the salt increases with increasing salt concentration. At high salt concentrations adding salt is likely to decrease the gas-phase ion intensity because the protein ions are neutralized by the counterions in the IR laser ablation nanodroplet. This decrease upon salt addition has been reported for lysozyme aqueous solution by Brutschy and coworkers.10 We also confirmed this experimentally. Figure 7

Figure 7. Gas-phase ion intensity isolated from 20 μM lysozyme solution as a function of NaCl concentration.

shows the gas-phase ion intensity isolated from 20 μM lysozyme solution as a function of the added NaCl concentration. The positive ion intensity in the gas phase decreases with increasing NaCl concentration, which can be interpreted by the neutralization of the positive lysozyme ion by Cl− ions in the nanodroplet. We also confirmed the model by observing positive ions produced from a 20 μM BSA + 250 μM NaOH solution. Figure 8 shows the gas-phase positive ion intensity as a function of

5. CONCLUSION Protein ions are isolated into the gas phase by an IR laser ablation of a droplet beam. We analyze the pH dependence of the gas-phase ion intensity according to a nanodroplet model. This work shows the general applicability of the nanodroplet model for the gas-phase isolation of protein molecules because the model describes the mechanism of the isolation of both positive and negative ions from solutions with a wide pH range. The model indicates that the protein molecules keep their properties in the solution during isolation in the gas phase by the IR laser ablation. Hence, the droplet-beam IR laser ablation method is shown to be applicable to studies on structures and dynamics of molecules in solutions in an isolated form in the gas phase.

Figure 8. Intensity of ions produced from a 20 μM BSA + 250 μM NaOH solution as a function of the NaCl concentration.

NaCl concentration. The ion intensity increases and decreases with increase in the NaCl concentration in the 0−50 and 50− 100 mM ranges, respectively. The increase for [NaCl] = 0−50 mM is explained by the nanodroplet model as described above. The decrease for [NaCl] = 50−100 mM is discussed in section 4.4. 4.4. Charge Inversion by Isolation of Ions into the Gas Phase. As shown in Figure 8, positive BSA ions are produced E


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AUTHOR INFORMATION

Corresponding Author

*(J.K.) Tel +81-3-3986-0221; Fax +81-3-5992-1029; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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Mechanism of Protein Molecule Isolation by IR Laser Ablation of

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