Protein Fragmentation Due to Slow Highly Charged Ion Impact

by
Christiane Ruehlicke ^{a, á}, Dieter Schneider ^a,
Markus Schneider ^a, Robert D. DuBois ^b, Rod Balhorn ^c

^a Lawrence Livermore National Laboratory,
Dept. of Physics and Space Technology,
Mail Stop: L-421, P.O. Box 808, Livermore, CA 94550
e-mail: ruehlicke1@llnl.gov, schneider2@llnl.gov

^b Department of Physics, University of Missouri-Rolla,
Rolla, MO 65401
e-mail: rdubois@physics.umr.edu

^c Lawrence Livermore National Laboratory,
Dept. of Biology and Biotechnology,
Mail-Stop: L-452, P.O. Box 808, Livermore, CA 94550
e-mail: balhorn2@llnl.gov

^á Christiane Ruehlicke is affiliated with
Universität Bielefeld (Bielefeld, Germany) through Rainer Hippler.

Abstract

We present first results of experiments on the fragmentation of biomolecules using highly charged heavy ions. Fragmentation and modification of oligopeptides, such as dimerization and attachment of ionic salt components, have been observed by means of mass spectrometry. Plasmid DNA molecules were imaged with an AFM after ion irradiation and profound molecular damage was found.

Introduction

Biomolecules such as proteins and DNA not only have important functions in our cells but they are also being engineered for a variety of applications in biotechnology. It is therefore desirable to find means to modify the molecular structure of peptides and DNA in a predictable and controlled way.

The attachment of ligands, polymerization and controlled fragmentation are effects that various groups are investigating via heavy ion irradiation. Fragmentation is also of interest because of its potential application to protein sequencing, an analytic process still limited by the availability of proteases, which are used to cut large proteins into peptide fragments for sequencing.

Until now, the investigation of heavy ion interaction with biomolecules has almost exclusively been done using fission fragments, typically with ions generated by the spontaneous fission of ²⁵²Cf. MacFarlane and Torgerson (1976) found that irradiation of peptides with such fission fragments leads to fragmentation at the sites of the peptide bonds, while Bouchonnet (1992) reported fragmentation of the sidechains in individual amino acids.

These heavy ions carry kinetic energies that are ca. three orders of magnitude higher than their potential or ionization energies. Therefore most of the interaction between target and projectile is assumed to be collisional rather than electronic. Highly charged ions (HCI) extracted from an electron beam ion trap (EBIT) usually have kinetic energies of a few 100 keV and they can be decelerated to much lower kinetic energies. This enhances the electronic interaction over the collisional one. For high ionization states (up to U⁹²⁺ ions have been produced in EBIT) the potential energies also exceed 100 keV, which can lead to new and exotic effects. For example experiments on highly charged ion interactions with solid surfaces have shown new effects, such as the emission of large clusters (up to mass m = 1000 amu for SiO₂, e.g.) and enhanced sputtering yields (Schneider and Briere, 1996). The emission of large numbers of electrons on a fs timescale due to single ion impact has been shown to yield up to a few 100s of electrons per incoming ion (McDonald, 1992). This may open up the possibility to study localized, hot electron plasma induced processes, that occur on a fs timescale. The formation of blisterlike surface defects due to single ion impact has been observed on mica using an atomic force microscope (AFM) (Ruehlicke, 1995). In this context potential applications of highly charged ions gain increasing interest also as techniques for ion beam focusing and steering are being improved.

2. Experimental

2.1. Target Preparation

Targets used in the time of flight measurements were the peptides RRRVAC and RVA as well as two of their constituent amino acids R and A. The amino acids were purchased from a commercial source (Sigma Chemical Co., St. Louis, MO) while the peptides were custom synthesized using a PS3 Peptide Synthesizer (Rainin Instruments) and purified by reversed-phase high performance liquid chromatography. Aliquots of the molecules ( 50 µl of a 10 mg/ml solution in water ) were deposited on flat gold disks and allowed to dry for 15 hours in the presence of a desiccant. The targets were then clamped onto a sample ladder and mounted in the experimental area within 24 hours following their preparation.

2.2 Time of Flight Spectrometry of Peptides

Time of flight secondary-ion-mass-spectrometry (TOF-SIMS) was performed in a high vacuum (10^-10 Torr) chamber equipped for surface analysis at the end of the EBIT extraction beamline. Highly charged ions were produced in EBIT, whose function as an ion trap/source has been explained in detail elsewhere (Schneider, 1991, Levine, 1989). These ions were then extracted to use as a projectile beam. Primary ions were Xe¹⁵⁺, Xe⁴⁴⁺, Xe⁵⁰⁺, Au⁶⁵⁺ and Th⁷⁰⁺, extracted at 7 kV * q, where q is the net ion charge with fluxes of ca. 1000 ions/s.

The TOF spectra were obtained using a TOF-SIMS spectrometer (Fig. 1) (Schneider and Briere, 1996). Secondary ions are accelerated between the target and a channelplate detector using voltages of a few kV. For negative secondary ions the start signal was taken from electrons emitted from the sample upon individual ion impact. For positive secondary ions protons provided the start signal. The stop signals were given by subsequently arriving secondary ions.

The flight time t of the secondary ions over a given distance and in a given electric field is related to the mass/charge ratio:

The positive secondary yield depends on the probability of proton emission, which might follow a different projectile charge state dependence than the larger mass secondary ions. However, this affects the secondary ion yield only if there is no proton emitted after primary ion impact.

2.3. DNA Irradiation

In order to obtain images of HCI induced damage of biomolecules, atomic force microscopy was performed on DNA molecules. 50 µl amounts of an aqueous solution of plasmid pUC18 DNA molecules (molecular mass ca. 50000 amu, Sigma Chemicals Co., St. Louis, MO) were deposited on muscovite mica surfaces and allowed to dry. The mica surfaces had been coated with 3-aminopropyltriethoxysilane (Bezanilla, 1995) to facilitate adhesion of the DNA on the surface.

After the unirradiated samples were imaged with an AFM they were then mounted on a sample ladder and placed in the ion extraction beamline at a vacuum of ca. 10-8 Torr. Xe⁴⁴⁺ ions were extracted from EBIT at 7 kV * q and directed onto the targets. Targets were irradiated for a few hours until a total fluence of 100 ions/µm² was achieved.

Upon removal from the vacuum system the samples were reimaged with the AFM. Measurements were made using a Park Scientific Instruments LS system in contact mode.

3. Results and Discussion

3.1. Time of Flight Spectra of Peptides

Results from the TOF measurements are shown in Figures 2 - 4. The large numbers of very low mass ions, e.g., H, C, and O compounds, do not originate solely from the molecular sample but also from contaminant species and the substrate and are common in these spectra regardless of the target material. These compounds and atomic ions of alkali metals and halides used during sample preparation dominate the spectra up to masses of ca. 100. Mass fragments that are unique to the peptide samples are observed in the higher mass range.

The positive secondary ion spectrum of the amino acid alanine (Fig. 2) shows a series of mass fragments that are both smaller and larger than the intact molecule. Most prominent are the mass contributions of the intact molecule as well as the molecule of twice the intact molecule mass. It is to be noted that this mass does not correspond to the dipeptide mass A-A, which would be 18 mass units below the double mass, since a water molecule must be released during the formation of the peptide bond.

In the case of the peptide RVA the positive spectrum (Fig. 3b) shows also a series of fragments, as well as a significant contribution at the intact molecule mass.

The occurrence of even higher masses suggests that the intact molecule combines with other fragments and possibly with Na, which is present in small concentrations in the peptide solution. The RVA spectra have been taken for two different charges of the same primary ion: Xe¹⁵⁺ has a potential energy of ca. 2 keV while Xe⁵⁰⁺ has 102 keV of potential energy. Thus the kinetic energy differs by a factor of ca. 3, given the dependence on the ion charge. For the same number of projectile ions hitting the target we have found that the sputter yield is at least one order of magnitude higher for the higher incident charge state. The Xe¹⁵⁺ spectrum shows virtually no masses larger than m = 100 amu, which indicates that higher masses are not ejected by impact of the lower charge ion or that their intensity is below the threshold limit. It should be noted that the analyzing electronics used in these experiments are not capable of recording multihits, therefore higher masses are blocked out preferentially.

In the negative RVA spectra (Fig. 3a) the same dependence of the secondary yields on the incident ion charge has been observed. The spectrum taken with the higher incident charge also shows a pronounced series of fragments with high intensities. These fragment series seem to be generally more pronounced in the negative spectra. A contribution at the intact molecule mass is observed as well.

We also studied the larger peptide RRRVAC, which released molecular fragments and compounds up to mass 1000 in the negative case (Fig. 4a). This is well beyond the mass of the intact molecule ( m = 763 u). The positive spectrum (Fig. 4b) also shows a fragment series, but it is much less distinct and does not extend into as high mass regions as the negative one.

3.2. Results of DNA Irradiation

AFM images reveal a profound structural change in the plasmid DNA due to HCI irradiation (Fig. 5). HCI impact has caused extensive fragmentation of the DNA molecules, to the point where the circular structure of the molecules is no longer recognizable. Some large molecular clusters have also formed which may result from breaking the bonds that keep the DNA attached to the mica surface, allowing multiple DNA molecules to aggregate or interact.

3.3. Discussion

The underlying mechanisms for breakup and ablation are not yet understood but it is assumed that a variety of processes occur, which originate in the large electric field induced by the high ion charge. This high field is likely to cause the removal of binding electrons, therefore causing the formation and ejection of molecular fragments. The electron depletion of the solid substrate by the HCI is also expected to be a factor in the ablation and ejection process, where weakening of the binding between the molecule and the surface precedes the desorption. Upon removal of binding electrons the molecule can change its structure and rearrange itself to form chemical species which would be otherwise unstable.

While the structure, binding and adhesion of the peptide molecules differs significantly from the solid surfaces, some of the responses to HCI impact are similar, e.g. the higher secondary ion yields and occurrence of high mass clusters due to HCI impact have been observed in sputtering solid surfaces with HCI as well (Schneider and Briere, 1996). The ring shaped circular pattern of the small fragments around clustered heavier fragments following the breakup of the DNA may indicate a Coulomb explosion type reaction.

4. Summary

The first results of fragmentation studies of biological molecules by HCI are presented. TOF-SIMS spectra of peptides and amino acids show the ablation of intact molecules as well as fragmentation of the molecules into a number of fragments. In the negative spectra a very pronounced series of molecular fragments has been found to occur, while the positive spectra exhibit more of a continuum of fragment peaks with lower relative intensities and which occur at lower masses than in the negative case. The ejection of molecules of twice the intact molecules' mass has been observed and the secondary sputter yield has been shown to increase significantly for high incident charge states. This is consistent with findings of HCI interaction with solid surfaces. In addition to the TOF-SIMS measurements of peptides plasmid DNA molecules were irradiated with HCI and the resulting dramatic molecular damage was visualized using an AFM.

5. References

Bezanilla M., Manne S., Laney D.E., Lyubchenko Y.L., et al., Langmuir, Feb. 1995, V 11 N 2: 655 - 659

Bouchonnet S., Denhez J.P., Hoppilliard Y., Mauriac C., Anal. Chem. 1992, 64, 743 - 754

Levine, M.A., Marrs R.E et al., Nucl. Instr. and Meth. in Phys. Res. B43, 1989, 431 -440

McDonald, J.W., Schneider D., Clark M.W. and Dewitt, D., Phys. Rev. Lett. 68, 1992, 2297

Ruehlicke C., Briere M.A. and Schneider D., Nucl. Instr. and Meth. in Phys. Res. B99, 1995, 528 - 531

Schneider D. M.W. Clark, Penetrante B.M., McDonald J., DeWitt D. and Bardsley J.N., Phys. Rev. A44 N5, 1991, 3119 - 3124

Acknowledgment

This work has been performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract No. W-7405-ENG-48.

á Christiane Ruehlicke is affiliated with Universität Bielefeld (Bielefeld, Germany) through Rainer Hippler.

Protein Fragmentation Due
to Slow Highly Charged Ion Impact