Fragmentation of Protonated Peptide Ions via Interaction with

Anal. Chem. 2006, 78, 3055-3061

Fragmentation of Protonated Peptide Ions via Interaction with Metastable Atoms Vadym D. Berkout*

MassTech, Inc., Columbia, Maryland 21046

Fragmentation of protonated peptide ions via interaction with low kinetic energy electronicallyexcited metastable argon atoms was studied in a linear trap-time-of-flight mass spectrometer. Metastable argon atoms were generated using a glow discharge-type source. Protonated peptide ions of substance P, bradykinin, fibrinopetide A, and insulin oxidized chain B were produced by electrospray ionization and trapped in a quadrupole ion guide for 100-400 ms. Intensive series of c- and z-ions were observed in all cases. The kinetic measurements of the fragmentation rates are consistent with calculations of the reaction cross section based on the Landau-Zener approximation. With the completion of the large-scale human DNA sequencing project, it is becoming increasingly important to obtain information at the proteome level for a more precise understanding of cell processes. Protein identification and characterization of their posttranslational modifications are some of the main areas that proteomics attempts to address.1,2 Tandem mass spectrometry (MS/MS) is currently one of the most important proteomics tools due to its high sensitivity (subfemtomole level) and specificity. The most prevalent MS/MS methods in use are based on collisioninduced dissociation (CID) of protonated peptide cations. In this process, which generally leads to cleavage of the peptide backbone amide bond to produce b-type and y-type sequence ions, peptides are kinetically excited and undergo multiple collisions with neutral gas molecules. The energy acquired in the collision is rapidly distributed throughout all covalent bonds. Fragment ions are formed when the internal energy exceeds the activation barrier required for a particular bond cleavage. The drawbacks of CID include the facile losses of labile groups involved in many important posttranslational modifications and incomplete backbone fragmentation in many cases.3 A technique that has been shown to complement CID of multiply protonated peptide cations is electron capture dissociation (ECD). Capture of a thermal electron by a protonated peptide is exothermic by ∼6 eV and causes fragmentation of N-CR bonds, yielding N-terminal c-fragments and C-terminal z-fragments4-6 In * E-mail: [email protected]. (1) Pandey, A.; Mann, M. Nature 2000, 405, 837-846. (2) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 5011-5015. (3) Simpson, R. J.; Connoly, L. M.; Eddes, J. S.; Pereira, J. J.; Moritz, R. L.; Reid, G. E. Electrophoresis 2000, 21, 1707-1732. (4) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. 10.1021/ac060069a CCC: $33.50 Published on Web 04/05/2006

© 2006 American Chemical Society

contrast to collision-induced dissociation, ECD is believed to be nonergodic;4 that is, the cleavage happens prior to any intramolecular energy redistribution. As a result, labile modification groups are preserved. ECD generally results in cleavage of a wider range of peptide backbone bonds than CID with less dependence on peptide composition.7 The maximum cross section for electron capture dissociation is observed for thermal energies.4 This is the main reason up-to-date ECD was successfully realized only in FTICR mass spectrometers, where the electric field is very weak, and additionally, the strong magnetic field confines electrons. The presence of strong (100-1000 V of amplitude) radio frequency (rf) electric fields in quadrupoles and ion traps makes difficult an introduction of low-energy electrons to the area where ions are located. So far, only the limited success was demonstrated.8,9 A new fragmentation techniqueselectron-transfer dissociation (ETD)sovercoming the technical challenges of introducing lowenergy electrons into strong oscillating rf fields, was recently proposed.10-12 In ETD, singly charged anions transfer an electron to the multiply protonated peptides in an ion trap and induce fragmentation of the peptide backbone along pathways that are similar to those observed in electron capture dissociation. Simultaneous trapping of cations and anions is readily accomplished by the rf quadrupole field. It has been noted,13 however, that the peptide structural information that can be obtained using ETD is charge-state dependent. The doubly charged peptide cations give much poorer sequence coverage than triply protonated cations, with fragmentation often limited to one or both ends of the peptide. It was also demonstrated14 that the percent of ETD process decreases with peptide size. (5) Zubarev, R. A.; Kruger, N.A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 28572862. (6) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N.A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (7) Zubarev, R. A. Mass Spectrom. Rev. 2003, 22, 57-77. (8) Baba, T.; Hashimoto, Y.; Hasegawa, H.; Hirabayashi, A.; Waki, I. Anal. Chem. 2004, 76, 4263-. (9) Silivra, O. A.; Kjeldsen, F.; Ivonin, I. A.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2005, 16, 22-27. (10) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528-9533. (11) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F. Int. J. Mass Spectrom. 2004, 236, 33-42. (12) Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 1020-1030. (13) Pitteri, S. J.; Chrisman, P. A.; Hogan, J. M.; McLuckey, S. A. Anal. Chem. 2005, 77, 1831-1839. (14) Pitteri, S. J.; Chrisman, P. A.; McLuckey, S. A. Anal. Chem. 2005, 77, 56625669.

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Fragmentation induced by electron transfer in high-energy collisions (100 keV) between doubly protonated peptide ions and Na or C60 was demonstrated.15,16 Further development of this technique was demonstrated in ref 17, where the authors used a beam from a fast bombardment (FAB) gun to fragment the peptide ions stored in a 3D ion trap. Fragmentation spectra were similar to the spectra observed using ECD. The authors hypothesized that the fragmentation patterns that were observed are due to the interaction of peptide ions with metastable, electronically excited species generated by the FAB gun.17 It is not clear, however, how the authors of ref 17 excluded secondary processes caused by collisions of fast (5 keV) neutral atoms with buffer gas and the internal metal surface of the 3D ion trap and why high-energy CID fragments were not observed in their experiments. In this study, we present direct evidence of electron transfer and the subsequent fragmentation of peptide ions during collisions with a low kinetic energy beam of metastable electronically excited argon atoms. The kinetic measurements of fragmentation rates and a model describing the dependence of the reaction cross section on the collision energy are also presented. EXPERIMENTAL SECTION Reagents. Solutions of peptides were prepared in molecular biology grade water (Cambrex Bio Science, Rockland, ME), reagent grade methanol, and glacial acetic acid (Sigma-Aldrich, St. Louis, MO) (2% in 1/1 water/methanol). Substance P, bradykinin, fibrinopeptide A, and insulin oxidized chain B were purchased from Sigma-Aldrich and were used without further purification. Mass Spectrometry. The time-of-flight mass spectrometer with orthogonal acceleration and an ESI ion source is shown schematically in Figure 1. It consists of an electrospray ion source, atmospheric pressure (AP) interface, octopole ion guide, a system of quadrupole ion guides, source of metastable atoms, and a timeof-flight mass analyzer. The AP interface consists of a heated capillary (0.4-mm i.d.) and a quadrupole ion guide (6.35-mm rod diameter). The pressure in the AP interface (∼1 Torr) was maintained by a 30 m3/h roughing pump. The octopole ion guide (3.2-mm rod diameter) was operated at ∼0.1 Torr pressure. Both, the quadrupole and octopole were driven by an rf generator, built in-house, according to the design described in ref 18. A capacitive divider allowed the application of different rf amplitudes to the quadrupole and octopole ion guides, respectively. The quadrupole and octopole rods were offset to some dc potential, applied through decoupling capacitors. A section of the quadrupole ion guides (8-mm rod diameter) was separated from the octopole by a 2-mm-diameter aperture. The quadrupoles were driven by a SRS (Sunnyvale, CA) model DS340 sine-wave signal generator coupled through an ENI (Rochester, NY) model 240L broadband rf power amplifier. An rf coupling transformer, built in-house, gave an output voltage 0-500 (15) Hvelplund, P.; Liu, B.; Nielsen, S. B.; Tomita, S.; Cederquist, H.; Jensen, J.; Schmidt, H. T.; Zettergen, H. Eur. Phys. J. D 2003, 22, 75-79. (16) Hvelplund, P.; Liu, B.; Nielsen, S. B.; Tomita, S. Int. J. Mass Spectrom. 2003, 225, 83-87. (17) Misharin, A. S.; Silivra, O. A.; Kjeldsen, F.; Zubarev R. A. RCM 2005, 21632171. (18) Jones, R. M.; Gerlich, D.; Anderson, S. L. Rev. Sci. Instrum. 1997, 68, 33573362.

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Figure 1. Schematic view of a linear trap-TOF mass spectrometer.

Figure 2. Schematic view of a glow discharge-type source for the production of a metastable atoms beam.

V0-p (zero-to-peak and pole-to-ground voltage) in the frequency range of 100 kHz-5 MHz. The transformer also provided the required 180° phase difference between the rod pairs. The quadrupole rods were offset to some dc potentials. The dc voltages applied to the entrance and exit apertures of the last quadrupole were changed by NAND gates with open collector outputs Texas Instruments (Dallas, TX) model SN74LS03 controlled by SRS model DG535 digital signal generator. A beam of metastable electronically excited argon atoms was introduced between the quadrupole rods. The gas, supplied to the source, created an expanding flow, which carried metastable atoms into the quadrupole ion guide. This low kinetic energy beam of metastable atoms interacted with peptide ions collimated along the central axis of the quadrupole ion guide, causing their fragmentation. A schematic of the metastable atoms beam source is shown in Figure 2. The glow discharge-type source that we used has an asymmetrical electric field between a radially separated cathode and anode.19 The asymmetrical field allows better separation of the neutral metastable atoms and the charged particles. A tapered rod (1.5-mm diameter) made from oxygen-free copper served as

the cathode. The anode was planar and located off-axis immediately after the aperture separating the two chambers. The use of a planar electrode for the anode increased stability of the discharge (by providing a larger surface to collect electrons). The discharge (I ≈ 5 mA, V ≈ 300 V) was initiated by applying 4.55.0 kV through a limiting 1.0 MΩ resistor. A negative potential was applied to the cathode, while the anode was grounded. The high-pressure chamber had a 0.5-mm-diameter exit aperture. The pressure in the first chamber (where the cathode is located) was ∼25 Torr, while the pressure in the quadrupole region was ∼5 mTorr. Two lenses, located downstream, were covered by nickel grids (BuckbeeMears, St.Paul, MN) with 70 wires/in. To prevent penetration of any remaining electrons into the quadrupole region, they were held at a few hundred volts negative potential. After passing through the final aperture (1-mm diameter), ions were focused by an Einzel lens into the pulser region of the timeof-flight mass analyzer. The time-of-flight analyzer chamber was pumped down by two Leybold (Export, PA) model Turbovac 361 350 L/s turbomolecular pumps to a pressure on the order of 10-7 Torr. Deflection plates shield the low-energy ion beam from the high voltages applied to the accelerator column and also make a final adjustment of the beam. Initially, the electric field in the gap of the pulser region is close to zero. The last grid of the extraction plates is held at a small positive potential (typically 6-12 V) to prevent field penetration20 from the accelerator column into the pulser region. When the pulser region is filled with ions, both push-out and draw-out voltage pulses are applied simultaneously to the extraction plates. The intermediate plate is kept at ground potential. The extraction pulses, produced by Behlke (Kronberg, Germany) model HTS 31GSM high-voltage push-pull switches, have typical amplitudes of ∼330 V, a rise time of 30 ns, and a duration of 3 µs. The duration of the pulse is chosen to guarantee that the heaviest ions have enough time to leave the pulser region. The values of the draw-out and push-out pulses are chosen to ensure a homogeneous electric field between extraction plates. This reduces ion scattering near the intermediate grid where defocusing effects could be strong for ions with a relatively low kinetic energy. After leaving the pulser region, ions are accelerated to energies of ∼5 keV by a uniform dc field in the acceleration column. The ions then move with constant velocities in the fieldfree drift region. The lengths of the pulser, acceleration, and fieldfree regions are 0.9, 4.6, and 48 cm, respectively. To keep the ion source and ion pulser potentials close to the ground, the drift region is floated at a high-voltage potential. A perforated metal cover prevents penetration of the ground potential of the vacuum chamber into the drift region, while allowing effective pumping of the inside volume. Having traversed the drift region, ions enter a single-stage electrostatic mirror, which was tuned together with a dual-stage accelerator to provide second-order space focusing on the initial ion position.21,22 The field-free region is separated (19) Bertrand, M. J.; Faubert, D.; Peraldi, O.; L′Heureux, A. J. Am. Soc. Mass Spectrom. 2001, 12, 754-761. (20) Dodonov, A. F.; Chernushevich, I. V.; Laiko, V. V. Electrospray Ionization on a Reflecting Time-of-Flight Mass Spectrometer. In Time-of-Flight Mass Spectrometry; Cotter, R. J., Ed.; ACS Symposium Series 549; American Chemical Society: Washington, DC, 1994; pp 108-123. (21) Mamyrin, B. A.; Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys. JETP 1973, 37, 45-53. (22) Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A. J. Mass Spectrom. 2001, 36, 849-865.

from the accelerator column and the electrostatic mirror by BuckbeeMears grids with 114 wires/in. having a transmission of 88.6%. The reflected ions pass back through the drift region before striking a detector. The detector was assembled using a Hamamatsu (Bridgewater, NJ) resistance matched pair of microchannel plates with a 12-µm channel diameter and a 12° bias angle. The front plate of the detector was at the same potential as the drift region (-5 kV), thus having the anode at high potential. The data acquisition system was decoupled from the high voltage by a ferrite transformer. The signal was amplified by an Ortec (Oak Ridge, TN) model 9327 1-GHz amplifier and recorded by a 100ps time digitizer (Ortec model 9353). When the ion signal was too intense for the time digitizer, a digital signal averager (Ortec model FastFlight-2) was used to record data. Voltages for the pulsers, drift region, and ion mirror were provided by Applied Kilovolts (Sussex, UK) models HP10 and HP5 stable, low-noise, high-voltage power supplies. RESULTS AND DISCUSSION Interaction of Metastable Argon Atoms with Different Gases. To prove that the above-described source generates a substantially neutral gas flow with electronically excited metastable argon atoms, the entrance capillary was closed and different gases were supplied into the first section of orthogonal chamber. This created a gas flow along the quadrupoles axis. The resulting pressure in the second section of the orthogonal chamber was ∼1 mTorr (background pressure in this section was less than 10-5 Torr). The frequency and amplitude of the rf voltage supplied to the quadrupole ion guide was tuned to transmit low m/z ions. Spectra, which were averaged over 10 s, are shown in Figure 3. Since both, ions and electronically excited atoms, are produced in the discharge, two different processes may have taken place:23

M + Ar•+ f M•+ + Ar M + Ar* f M•+ + Ar + e

The first process describes charge exchange ionization and the second, Penning ionization. Recombination energy of Ar ions equals 15.76 eV, and the energy of the two most populated metastable states 3P0 and 3P2 equal 11.72 and 11.55 eV, respectively.24 Ionization potentials of nitrogen, methane, ammonia, and isobutane equal 15.58, 12.61, 10.07, and 10.68 eV, respectively.24 Energetically, charge exchange ionization is possible for all studied gases, but practically no nitrogen ion signal was recorded and only a weak methane ion signal was observed. Ionization of methane could also take place in interaction with some higher lying electronically excited states of argon atoms. The ionization potential of ammonia is ∼1.5 eV lower that argon 3P0 and 3P2 metastable levels. A strong molecular radical cation signal was recorded (∼2 orders of magnitude larger than found for methane) in this case. An intense spectrum, consisting mainly of fragment ions, was also observed when isobutane was supplied. This indicates that the argon beam entering the quadrupole ion guide (23) Harrison, A. G. Chemical Ionization Mass Spectrometry, 2nd ed.; CRC Press: Boca Raton, FL, 2000. (24) NIST Chemistry WebBook, http://webbook.nist.gov/chemistry/.

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Figure 3. Ionization spectra of nitrogen, methane, ammonia, and isobutane produced in interaction with metastable electronically excited argon atoms.

consists mainly of neutral particles and contains substantial amounts of metastable electronically excited states 3P0 and 3P2. Small amounts of argon ions initially present in the beam will be ejected from the quadrupole ion guide in peptide fragmentation experiments, since low m/z cutoff was set to ∼250 in these experiments. Fragmentation of Peptides in Trapping Mode. Peptide ions were trapped in the last quadrupole for 100-400 ms to increase the time available for interaction with electronically excited metastable argon atoms. Ions were produced by electrospray from 10 µM solutions of peptides. Injection time into the quadrupole linear trap was 1-5 ms. Argon gas was flowing through the source during experiments, and the presence of metastable electronically excited atoms in the argon beam was controlled by turning the discharge on and off. Typically, data were acquired for 30 s. The spectrum for substance P, fragmented in interaction with low kinetic energy metastable argon beam, is shown in Figure 4. The spectrum contains a dominating series of c-fragment ions: singly charged from c4 to c10 and doubly charged from c8 to c10. Ion signals at m/z ) 355, 371, 391, 413, 653, and 803 are due to the contaminants. A strong b10(2+) ion (m/z ) 600.3), corresponding to methionine loss from C-terminal, was also observed when the discharge was off. It was also observed in substance P electrospray spectrum on LCQ ion trap mass spectrometer (Finnigan, San Jose, CA) and is, presumably, present in the sample itself. The isotopic structure of m/z ) 674 doubly charged and m/z ) 1349 singly charged ions was different when the discharge was on. The intensity of the second isotope was larger than for normal isotopic distribution. This indicates the presence of [M + 3H]•2+ and [M + 2H]•+ ions produced via charge reduction. 3058

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Figure 4. Fragmentation spectrum of substance P obtained via interaction with a low kinetic energy beam of metastable argon atoms (* denotes contaminant peaks).

The spectrum of bradykinin fragmented in interaction with metastable argon atoms is shown in Figure 5. The spectrum shows nearly complete series of c- and z-ions (with the exception being the bond N-terminal to the proline residue, which is not susceptible to cleavage by ECD7). Some aand y-ions were also observed. The presence of [M + 3H]•2+ was even more profound than in the case of substance P. [M + 2H]•+ was also present in the spectrum. It is interesting to note that fragmentation in interaction with metastable argon atoms produce more c- and z-fragments than ETD.14 To study fragmentation of larger peptides via interaction with low kinetic energy metastable argon beam, fibrinopeptide A (mw

Figure 5. Fragmentation spectrum of bradykinin obtained via interaction with a low kinetic energy beam of metastable argon atoms (* denotes contaminant peaks).

Figure 6. Fragmentation spectrum of fibrinopeptide A obtained via interaction with a low kinetic energy beam of metastable argon atoms (* denotes contaminant peaks).

Figure 7. Fragmentation spectrum of insulin oxidized chain B obtained via interaction with a low kinetic energy beam of metastable argon atoms (* denotes contaminant peaks).

1536.6) and insulin oxidized chain B (mw 3496.0) were chosen. The corresponding spectra are shown in Figures 6 and 7, respectively. The intensities of c- and z-ions are reduced compared to molecular ions, but still easily identifiable. Intense y-ions, both singly and doubly charged, are produced in the case of fibrinopeptide A fragmentation. Fragmentation of multiply charged

Figure 8. Dependence of the number of substance P fragment ions produced via interaction with metastable argon atoms on the interaction time.

insulin oxidized chain B peptide in interaction with metastable argon atoms produces also doubly charged c- and z-ions. Y-ions are considerably less noticeable in this spectrum in comparison with fibrinopeptide A fragmentation spectrum. Kinetic Measurements. To study the dependence of peptide cation fragmentation on interaction time with metastable argon atoms, the dependence of ion signal on trapping time was measured. Substance P was studied in these experiments. Since data were recorded by the time digitizer, the number of counts is directly proportional to the number of ions striking the detector (on condition of small ion intensities). The intensity dependence of parent and c-type fragments on interaction time is shown in Figure 8. From the data presented in Figure 8, we can conclude that the number of molecular doubly charged ions reduces slower than the number of triply charged molecular ions. The ratio of first and second isotopes of a doubly charged ion reduces with time, revealing the formation of [M + 3H]•2+. The number of c4 and c5 ions increases monotonically and linearly with time during first 150 ms. The number of c92+ and c102+ ions increases up to 300 ms and then starts to decrease. Interaction of selected ions with metastable argon atoms was studied with bradykinin (3+) molecular ion. Mass selection was performed in the first of three quadrupoles by applying (dc voltage to corresponding rod pairs. The mass selection window was ∼30 Da because the selection quadrupole was short (∼3 in.) and the pressure was relatively high (∼20 mTorr). The resulting spectrum, averaged over 30 s, is shown in Figure 9. Without the flow of metastable argon atoms, the spectrum in this region showed only noise, not exceeding 1 count for the same acquisition time. The inset demonstrates the formation of [M + 3H]•2+. The total intensity of c- and z-ions constitutes ∼1.0% of intensity of the [M + 3H]3+ ion. These data show the potential of this method for fast fragmentation of peptide cations. Reaction Mechanism. The electron transfer in the reaction MHnn+ + Ar*f MHn(n-1)+• + Ar+ can be considered in the Landau-Zener approximation,25,26 which is based upon a first(25) Landau, L. D. Phys. Z (USSR) 1932, 2, 46-51. (26) Zener, C. Proc. R. Soc. London 1932, A136, 696-702.

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Figure 9. Fragmentation spectra of bradykinin [M + 3H]3+ ion obtained via interaction with metastable argon atoms in continuous mode.

Figure 11. Dependence of cross section of the electron transfer from electronically excited Ar(3P2) to a doubly charged peptide cation on the collision energy.

order perturbation treatment of a two-state model where there is interaction only near the crossing point of adiabatic potential curves corresponding to MHnn+ + Ar* and MHn(n-1)+• + Ar+ states (see Figure 10). Near the crossing point, a nonadiabatic transfer can take place from one state to another. The cross section of such transfer is defined by27

units, i.e., energies in units of 2IH ) 27.2 eV, and distances in units of a0 ) 0.529 Å). At large internuclear separations, the induced dipole polarization potential of the reactants (-Rd/2R4) may be neglected compared to Coulomb repulsion of the products, and we find derivatives of potentials are determined by

σ12 ) 4πRc2[1 - V11(Rc)/E]G(λ)

|V′11(Rc) - V′22(Rc)| ) (n - 1)e2/Rc2

(2)

Rc ) (n - 1)e2/(EA - I)

(3)

(1)

and also

where

G(λ) )

∫

∞

1

exp(-λx)

(1.0 - exp(-λx)) x

3

dx

and

2πH122 λ) pv[1 - V11(Rc)/E]1/2|V′11(Rc) - V′22(Rc)|

H12 is the coupling matrix element that determines the strength of electronic coupling between two states,V′11(Rc) and V′22(Rc) are the first derivatives of potentials at crossing point, v is incident velocity, and E is corresponding energy (all values are in atomic

where EA is an electron affinity of the multiply charged peptide ion and I is the ionization potential of the electronically excited argon atom. Of all parameters relevant to Landau-Zener approximation, the coupling matrix element H12 is the most difficult to evaluate. However, an approximate model can provide some insights into qualitative effects of experimental variables on the cross section of electron-transfer reaction. A semiempirical correlation for coupling matrix element was derived in27

H12* ) Rc* exp(-0.86Rc*)

(4)

where

Rc*) Rc/2{(2I)1/2 + (2EA)1/2} and H12*) H12/(I‚EA)1/2

Basic to this correlation is the assumption that the wave functions are nearly hydrogenic and that the particles are far enough apart so that the electron transfer is determined in a simple way by consequent tunneling. The dependence of the cross section of the electron transfer from electronically excited Ar(3P2) to a doubly charged peptide cation on the collision energy is shown in Figure 11. Figure 10. Potential energy diagram for interaction of peptide cation with metastable electronically excited atom. 3060

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(27) Olson, R. E.; Smith, F. T.; Bauer, E. Appl. Opt. 1971, 10, 1848-1855.

It was assumed that EA is 6 eV.28 Ionization potential and polarizability of metastable Ar(3P2) equal 4.2 eV24 and 323,29 respectively. The crossing distance equals 8.0 Å or 15.12 in atomic units for these parameters. The reaction cross section reduces when colliding energy increases from 0.03 to 0.1 eV, and then it remains constant up to ∼1 eV. At large energies it decreases ∼(ln E)-1/2. This dependence demonstrates that low kinetic energies of metastable argon atoms are preferable for the electrontransfer reaction with peptide cations. The source of the metastable argon atoms, described in the previous section, typically generates 1015 metastables s-1 srad-1.19 Metastable species effuse into the lower pressure region of the ion guide with thermal velocities (∼3 × 104 cm/c). For a characteristic distance of 2 cm, between the exit of the source and the multiple ion guide central axis, this translates into a concentration of metastable atoms on the axis of the ion guide (28) Breuker, K.; Oh, H.; Lin, C.; Carpenter, B.; McLafferty, F. W. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14011-14016. (29) Christophorou, L. G.; Illenberger, I. Phys. Lett. A 1993, 173, 78-84.

n* of the order of 1010 metastables/cm3. The reaction cross section for the electron transfer from metastable atom to a multiply charged peptide cation σ is of the order of 10-14 cm2 according to Figure 10. Based on this we can estimate the effective length for the interaction with metastable argon atoms taking place as 1/n*σ ∼ 104 cm, which corresponds to a time ∼100 ms that ions need to spend in a linear trap to be fragmented via interaction with metastable argon atoms. ACKNOWLEDGMENT The author thanks Dr. Andrey Vilkov for his assistance in atmospheric pressure interface design and Dr. Vladimir Doroshenko for helpful discussions.

Received for review January 10, 2006. Accepted March 8, 2006. AC060069A

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