Anal. Chem. 1996, 68, 463-472
Pulsed Gas Introduction for Increasing Peptide CID Efficiency in a MALDI/Quadrupole Ion Trap Mass Spectrometer Vladimir M. Doroshenko and Robert J. Cotter*
Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
A pulsed valve was used for studying the effects of introducing heavy gases at different stages of operation of a quadrupole ion trap and for increasing the efficiency of collision-induced dissociation (CID) of peptide ions at low values of the Mathieu parameter qz. When amounts of heavy gases comparable to that of the helium buffer gas were introduced during the ion trapping, ion isolation, and mass spectral recording stages, the effects on performance were generally small or negative. However, injection of heavy gases during CID provided considerable improvement in fragmentation efficiency that depended upon the particular gas used, its mass and pressure, and the amplitude of the excitation voltage. Efficient peptide fragmentation could be demonstrated for values of qz as low as 0.05, which permitted trapping of low-mass product ions and (in many cases) full recovery of the amino acid sequence. In this report, examples are provided of monoisotopic tandem mass spectra of peptide ions with masses up to 1570 Da. Since its initial introduction in 1968 as a method for increasing the fragmentation of metastable ions,1,2 collision-induced dissociation (CID) has been widely used to provide structural analysis in tandem mass spectrometers. These include quadrupole ion traps3 which provide the opportunity for multiple tandem (MSn) experiments.4 A distinctive feature of ion trap mass spectrometers (ITMSs) is the presence of helium buffer gas (at relatively high pressures of ∼1 mTorr) that serves to damp the ion motion5 and results in increases in both mass resolution and sensitivity. Thus, collisions with helium atoms are commonly (and conveniently) used as well for fragmentation following additional excitation of the analyte ions. Although CID has been successfully applied to the fragmentation of peptides in the ITMS,6 practical use of this instrument for amino acid sequencing is dependent on the ability to trap ions over a broad range of m/z. In an ITMS, the range of stable ions confined in the trap depends upon the dimensions of the trap, the magnitude of the direct current (dc) voltage, and the amplitude and frequency of the fundamental radio frequency (rf) voltage on (1) Jennings, K. R. Int. J. Mass Spectrom. Ion Phys. 1968, 1, 227-235. (2) Haddon, W. F.; McLafferty, F. W. J. Am. Chem. Soc. 1968, 90, 4745-4746. (3) Louris, J. N.; Cooks, R. G.; Syka, J. E. P.; Kelley, P. E.; Stafford, G. C.; Todd, J. F. J. Anal. Chem. 1987, 59, 1677-1685. (4) March, R. E. Int. J. Mass Spectrom. Ion Processes 1992, 118/119, 71-135. (5) Stafford, G. C.; Kelley, P. E.; Syka, J. E. P.; Reynolds, W. E.; Todd, J. F. J. Int. J. Mass Spectrom. Ion Processes 1984, 60, 85-98. (6) Kaiser, R. E.; Cooks, R. G.; Syka, J. E. P.; Stafford, G. C. Rapid Commun. Mass Spectrom. 1990, 4, 30-33. 0003-2700/96/0368-0463$12.00/0
© 1996 American Chemical Society
the ring electrode, expressed in terms of the Mathieu parameters az and qz, respectively.4 In the mass-selective instability mode of operation (where az ) 0), the low-mass cutoff depends only upon the fundamental rf voltage (i.e., qz) and may preclude trapping of low-mass ions formed by collisional excitation. Thus, in order to observe these ions and record a full product ion mass spectrum, CID should be carried out using the smallest possible values of qz. In practice, there is trade-off between this requirement and the need for high internal energy deposition to achieve effective fragmentation into different reactive channels. In particular, the maximum collision energy depends on the maximum kinetic energy, Ek, of excited ions, which in a pseudopotential well approximation at qz < 0.4 (ref 7) can be estimated as
Ek ) mω2r02/4 ) mqz2r02Ω2/32
(1)
where m is the ion mass, ω is the radial frequency of the dipole excitation field, Ω ) 2π(1.1) MHz is the radial frequency of the fundamental rf field, and r0 ) 1 cm is the radius of the ring electrode of a standard ITMS. As can be seen from eq 1, the maximum kinetic energy of an ion drops quickly upon lowering the parameter qz, and practical values of qz between 0.2 and 0.6 have generally been chosen to achieve high fragmentation efficiencies.3,6,8,9 CID experiments with helium as a buffer gas and qz < 0.1 result in ejection of the parent ions during axial excitation, since the trapping field strength is now considerably reduced, without production of fragment ions.3 The smallest values of qz (0.074) have been used successfully for the fragmentation of weakly bound Cs(CsI)n+ cluster ions,10 which required considerably lower dipolar excitation ω. A novel ion excitation method has been proposed that occurs near the boundary βz ) 0 (where βz ) 2ωz/Ω and ωz is the resonance or secular frequency of ion motion) of the stability diagram. Referred to as “boundary” activation, it has been reported to increase the allowable mass difference between parent and fragment ions.11 However, the gain in mass range (10-20%) is small for a complicated mode of operation that requires linking the rf and dc voltages on the ring electrode to prevent ion loss during excitation. (7) Major, F. G.; Dehmelt, H. G. Phys. Rev. 1968, 179, 91-107. (8) Cox, K. A.; Williams, J. D.; Cooks, R. G.; Kaiser, R. E. Biol. Mass Spectrom. 1992, 21, 226-241. (9) Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1995, 67, 2180-2187. (10) Morand, K. L.; Cox, K. A.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1992, 6, 520-523. (11) Paradisi, C.; Todd, J. F. J.; Vettori, U. Org. Mass Spectrom. 1992, 27, 12101215.
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Figure 1. Timing diagram of the experiment for studying the injection of argon at different stages of ion trap operation: (I) trapping of the ions; (II) ion isolation; (III) excitation and CID of remaining ions; (IV) analytical scan to produce the mass spectrum.
Table 1. Efficiency of CID Process for Injection of Different Gases into the Ion Trap Chambera gas injected into the trap chamber qz for CID
no gas injection
He
NH3
air
Ar
Xe
0.350 0.162 0.078
low no CID no CID
high low no CID
high high no CID
high high low
high high high
high high high
a Note that the stationary partial pressure of injected gas approximately corresponds to the pressure of helium buffer gas (∼0.5 mTorr).
Ultimately, energy deposition in CID processes depends upon the relative collision energies of the ion and the neutral gas. Thus, in addition to the ion kinetic energy, as expressed in eq 1, it also depends upon the relative masses of the ion, m, and the neutral target, M. In the ITMS, activation occurs in a stepwise fashion via multiple collisions,12 where the relative collision energy (Erel) in the center-of-mass frame (for m . M) is
Erel )
M E ≈ Mqz2r02Ω2/32 m+M k
(2)
for each collision. Thus, for heavy ions encountering a light target, the energy transferred in collisions depends almost exclusively upon the mass of the neutral target M, and the increase in energy deposition with increasing target mass is well known for the lowenergy CID experiments utilized in tandem quadrupole instruments.13 In addition, the effects of adding heavy target gases on 464
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low-energy CID efficiency and overall performance have been investigated for ion trap mass spectrometers,10,14-20 with higher activation fragmentation channels observed with use of argon, xenon, and other heavy gases. At the same time, trapping efficiency decreases as the heavy gas content in the buffer gas mixture is increased, so that an optimal mixture is obtained when the heavy gas content is only a few percent.10 The pressure of the buffer gas also affects the fragmentation pattern although its influence on sensitivity seems to be more important.15,16,20 Alternative methods for increasing energy deposition have been suggested, in particular surface-induced dissociation (SID).21 In all previous studies of the effects of heavy collision gases in the ITMS, target gas mixtures were utilized throughout all of (12) Brodbelt, J. S.; Kentta¨maa, H. I.; Cooks, R. G. Org. Mass Spectrom. 1988, 23, 6-9. (13) Nystrom, J. A.; Bursey, M. M.; Hass, J. R. Int. J. Mass Spectrom. Ion Processes 1983/1984, 55, 263-274. (14) McLuckey, S. A.; Glish, G. L.; Asano, K. G. Anal. Chim. Acta 1989, 225, 25-35. (15) Gronowska, J.; Paradisi, C.; Traldi, P.; Vettori, U. Rapid Commun. Mass Spectrum. 1990, 4, 306-313. (16) Glish, G. L.; McLuckey, S. A.; Goeringer, D. E.; Van Berkel, G. J.; Hart, K. J. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 19-24, 1991; pp 536-537. (17) Paradisi, C.; Todd, J. F. J.; Traldi, P.; Vettori, U. Org. Mass Spectrom. 1992, 27, 251-254. (18) Morand, K. L.; Hoke, S. H.; Eberlin, M. N.; Payne, G.; Cooks, R. G. Org. Mass Spectrom. 1992, 27, 284-288. (19) Nourse, B. D.; Cox, K. A.; Morand, K. L.; Cooks, R. G. J. Am. Chem. Soc. 1992, 114, 2010-2016. (20) Charles, M. J.; McLuckey, S. A.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1994, 5, 1031-1041. (21) Lammert, S. A; Cooks, R. G. J. Am. Soc. Mass Spectrom. 1991, 2, 487-491.
Figure 2. Monoisotopic CID mass spectrum of protonated molecules of dermorphin (m/z 803.37) at qz ) 0.162, using collisions with air molecules at a pressure of ∼3.5 mTorr and amplitude of SWIFT pulses applied between the end-cap electrodes for CID activation equal to (a) 0.94, (b) 1.26, (c) 1.58, (d) 2.2, and (e) 3.16 V.
the stages of ion trap operation, so that conclusions as to the effects of heavy gas addition on each stage were made from observations of the total effects on ITMS performance. The need to elucidate the distinct effects upon each stage of operation, as well as the desire to address the problem of increasing energy deposition at low qz, motivated the present study, in which heavy target gases were introduced separately at each stage using a pulsed valve. Pulsed valves have been used successfully to increase CID efficiency in the Fourier transform mass spectrometer (FTMS) in order to maintain ultra-high-vacuum conditions.22 In addition, pulsed gas introduction techniques have been utilized on the ITMS for chemical ionization and for ion-molecule reaction studies.23,24 In this work, the introduction of heavy target gases using a pulsed valve is combined with other methods for improving ITMS
performance that have been reported previously by researchers from our laboratory. These include methods for increasing the trapping efficiency of ions formed by the matrix-assisted laser desorption/ionization (MALDI) technique,25 for linearization of mass calibration of the instrument in the resonance ejection mode of operation,26 and for unit resolution parent ion isolation in the range of m/z over 1000 using stored waveform inverse Fourier transform (SWIFT) techniques.27 This combination of techniques has allowed us to conduct high-performance tandem experiments in which monoisotopic species of the parent peptide ion cluster are preisolated and the masses of CID fragment ions are assigned with accuracies of 0.2-0.3 Da. These experiments can be carried out at values of qz as low as 0.05 so that (in many cases) product ions corresponding to cleavages for all amide bounds can be trapped and recorded.
(22) Carlin, T. J.; Freiser, B. S. Anal. Chem. 1983, 55, 571-574. (23) Emary, W. B.; Kaiser, R. E.; Kentta¨maa, H. I.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 1990, 1, 308-311. (24) Einhorn, J. E.; Kentta¨maa, H. I.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 1991, 2, 305-313.
(25) Doroshenko, V. M.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1993, 7, 822-827. (26) Doroshenko, V. M.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1994, 8, 766-776. (27) Doroshenko, V. M.; Cotter, R. J. Rapid Commun. Mass Spectrom., in press.
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Figure 3. Monoisotopic CID mass spectrum of protonated molecules of dermorphin (m/z 803.37) at qz ) 0.162 using collisions with ammonium molecules at a pressure of ∼3.5 mTorr and amplitude of SWIFT pulses applied between the end-cap electrodes for CID activation equal to (a) 0.94, (b) 1.26, (c) 1.58, (d) 2.2, and (e) 3.16 V.
EXPERIMENTAL SECTION Instrumentation. Experiments were carried out on a modified Finnigan MAT (San Jose, CA) ion trap detector (ITD), which was previously modified for MALDI operation.9,25-28 The instrument utilizes the fourth harmonic (λ ) 266 nm) output from a Quantel International (Santa Clara, CA) Nd-YAG laser that illuminates a sample probe inside the ion trap, through the gap between the ring and end-cap electrodes. As described previously, a ramped trapping voltage method was used for efficient trapping of MALDI ions,25 in which the trapping rf field is increased during the ion flight into the cell. A modification of this method enables cumulative trapping of ions from several laser shots27 prior to the stages of monoisotopic ion isolation, excitation, and spectral recording. The stationary helium pressure in the trap itself has been estimated to be from 4 × 10-4 to 5 × 10-4 Torr.26 To accommodate pulsed valve operation, the ion trap assembly was remounted in a newly constructed two-section vacuum coffin (28) Doroshenko, V. M.; Cornish, T. J.; Cotter, R. J. Rapid Commun. Mass Spectrom. 1992, 6, 753-757.
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chamber, with the trap section pumped by a 330 L/s turbomolecular pump. At this pumping speed, the time for pumping out the trap section (measured on an oscilloscope from the output of an ion gauge) was generally
