Article pubs.acs.org/JPCA
Charge State of Lysozyme Molecules in the Gas Phase Produced by IR-Laser Ablation of Droplet Beam Jun-ya Kohno,* Kyohei Nabeta, and Nobuteru Sasaki Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan ABSTRACT: Molecules exhibit their intrinsic properties in their isolated forms. Investigations of isolated large biomolecules require an understanding of the detailed mechanisms for their emergence in the gas phase because these properties may depend on the isolation process. In this study, we apply droplet-beam laser-ablation mass spectrometry to isolate protein molecules in the gas phase by IR-laser ablation of aqueous protein solutions, and we discuss the isolation mechanism. Multiply charged hydrated lysozyme clusters were produced by irradiation of the IR laser onto a droplet beam of aqueous lysozyme solutions with various pH values prepared by addition of sodium hydroxide to the solution. The ions produced in the gas phase show significantly low abundance and have a lower number of charges on them than those in the aqueous solutions, which we explained using a nanodroplet model. This study gives quantitative support for the nanodroplet model, which will serve as a fundamental basis for further studies of biomolecules in the gas phase.
1. INTRODUCTION The function of biological molecules emerges in aqueous solutions in cooperation with adjacent molecules including the water solvent. For example, protein molecules assume their inherent folded structure in aqueous solution, which determines their biological properties.1 Protein molecules comprise a long peptide backbone chain with protruding side chains. Some of these side chains may be nonpolar and hydrophobic, while others may be negatively or positively charged. Polar amino acid side chains tend to gather on the exterior of the protein, where they can interact with water; nonpolar amino acid side chains tend to locate inside the protein to form a tightly packed hydrophobic core of atoms that are protected from unfavorable interactions with the water solvent. Thus, interactions between the amino acid side chains and the solvent water molecules determine the folded structure of the protein molecule. In addition, protein function is also influenced by interactions with adjacent water molecules, because the binding of functional molecules to the protein surface competes with solvent interactions. Therefore, it is important to study interactions of proteins and water molecules. The molecular function of the water molecules can be explored by comparing properties in isolation to those in the presence of water. In such studies, it is important to isolate the hydrated biomolecules in the gas phase under conditions which maintain the original solvation environment. While various isolation techniques have been developed to introduce biomolecules to the gas phase without suffering significant fragmentation, such as electrospray ionization2−5 and matrix-assisted laser desorption/ionization,6,7 we have employed IR-laser ablation of aqueous solutions in a vacuum in order to isolate the molecules (including biomolecules) into the gas phase directly from solution in order to retain the solutionphase properties. In this technique, a liquid sample is introduced into a vacuum environment and is irradiated with © 2012 American Chemical Society
an IR laser having a wavelength resonant with the OH stretching mode of liquid water. The technique enables us to investigate the molecules as they exist in the condensed-phase environment, because the molecules are likely to be ejected from the solution into the gas phase within several microseconds after the IR-laser irradiation. This method was explored by Brutschy and co-workers, who developed a liquid beam−IRlaser desorption technique for the gas-phase isolation of biomolecules8,9 and extended its use to smaller liquid droplets, reducing sample consumption, which is invaluable for biomolecular studies.10−13 Abel and co-workers have also extensively studied isolation of biomolecules.14−16 We have also performed isolation of molecules and ions to investigate their structure and dynamics in solution.17−22 For example, the hydrophobic nature of the concentrated aqueous solution of arginine was determined based on the mass distribution of the isolated arginine ions and their hydrated clusters.23,24 Recently, we have developed a droplet-beam laser-ablation mass spectrometry (DB-LAMS) apparatus which enables us to deal with small amounts of biomolecular samples by introducing micrometer-sized droplets into a high vacuum environment. We confirmed that the droplet exists in the liquid phase in a vacuum,25 and we investigated the proton-transfer reaction of gas-phase isolated lysozyme molecules induced by UV laser irradiation.26 Further instrumental development is in progress, where the isolated ions are trapped for long times in a quadrupole electric field in order to investigate their properties.27 Along with the isolation studies, the mechanism for IR-laser ablation has been explored. Brutschy and co-workers proposed a “lucky survivor” model.28,29 They consider that a plume with a Received: September 28, 2012 Revised: December 11, 2012 Published: December 12, 2012 9
dx.doi.org/10.1021/jp3096506 | J. Phys. Chem. A 2013, 117, 9−14
The Journal of Physical Chemistry A
Article
region. We have observed protein ions (m/z ∼ 66 000) in the previous experiment,26 that are larger than [Lys]1+ (m/z ∼ 14 400) with maximal m/z in the present experiment. Accordingly, the deterioration was not taken into account in the analysis of the ion intensities.
supercritical phase emerges by irradiation of the IR laser on the liquid. Ions are liberated from the solution into the gaseous plume, where they suffer from recombination with counterions simultaneously produced in the plume. Thus, only a small amount of the initial ions can survive to become product ions. Baer and co-workers applied this model to interpret the abundance of ions produced by irradiation of an IR laser onto the solution.30 On the other hand, Abel and co-workers have proposed a different model, a nanodroplet model.31 Their model assumes that liquid IR-laser ablation produces liquid droplets over a wide range of diameters, where only nanosized droplets, including the solute ions of interest, result in formation of product ions by water evaporation from the nanodroplets. In this paper, we discuss the gas-phase abundance and charge distributions of multiply charged lysozyme ions isolated in the gas phase by IR-laser ablation, in comparison with those in aqueous solutions with varying pH. The results can be described by a nanodroplet model semiquantitatively.
3. RESULTS Figure 1 shows mass spectra of ions obtained from aqueous solutions of (Figure 1a) 20 μM Lys and (Figure 1b) 20 μM Lys
2. EXPERIMENTAL SECTION A detailed description of DB-LAMS has been given previously.25 The apparatus and the experimental procedures employed in the present study are described briefly. 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 an aqueous solution of 20 μM lysozyme (Lys) and x μM sodium hydroxide (NaOH) (x = 0−1000) was injected into air from the nozzle. Commercially available Lys (Seikagaku Biobusiness) and NaOH were used without further purification. The Lys reagent was supplied as the hydrochloride powder with a 2.1% Cl content. Aqueous solutions of Lys and NaOH were prepared by mixing amounts of the stock Lys and NaOH aqueous solutions, followed by subsequent dilution with deionized and distilled water. Gas flow from the inlet aperture to the first vacuum chamber carried the droplet through the second chamber to the third, where the droplet was admitted into the acceleration region of the TOF-MS. Multiply charged Lys ions were produced from the droplet by irradiation of an IR laser (3586 cm−1,
