Anal. Chem. 1992, 64, 2628-2635
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Tandem Mass Spectrometry of Peptides Using a Magnetic Sector/Quadrupole Hybrid-The Case for Higher Collision Energy and Higher Radio-Frequency Power Caroline D. Bradley, Jonathan M. Curtis,' and Peter J. Derrick Institute of Mass Spectrometry and Department of Chemistry, University of Warwick, Coventry CV4 7AL, England
Barry Wright Kratos Analytical, Barton Dock Road, Urmston, Manchester M31 2LD, England
Uslng a magnetlc sector/quadrupole hybrld tandem mass spectrometer dedgned for hlgh tranmlsslon at hlgh Ion energks (400-500 eV), lt has been poorlble to obtaln tandem mass spectra results for hlghmass Ions ( m / r 1000-2500) whlch resemble those achleved wlth four-sector tandem Instruments. Wlh a nwnber of peptldeIons, the hlgh laboratory cdlldon energks (at 400-500 eV compared to 30 eV) led to the observation of fragmentations which were not present at lower energies. At constant center-of-mass collldon energy, changingthe collklongas from Ar to He and at the same tlme changlng laboratory collldon energles from 400 to 4000 eV, resulted In more high-energy fragmentatlon pathways. Thls Implles a greater uptake ol Internal energy wlth He colllslon gas. I n the case of dynorphlnA fragment 1-9, a tandem mass spectrum was obtalned wlth the hybrld Instrument using 500 pmol of sample whereas a prevlous attempt had falled to obtaln usefulfragmentationby colllrlonal actlvatlonIn a hybrld Instrument.
INTRODUCT10N
Much of the interest in tandem mass spectrometry stems from the development of ionization methods for compounds with molecular masses in the kilodalton range and consequent applications of mass spectrometry in the broadest sense to biological areas. It is usual for a state-of-the-art doublefocusing mass spectrometer to have an mlz range extending up to 10 000-15 OOO, but four-sector mass spectrometers have not been useful for tandem mass spectrometry beyond masses of about 3 or 4 kDa. This inability to date to exploit the full mass range is a consequenceof,among other things, difficulties associated with fragmenting large ions. The limitations of hybrids in this regard have been yet more serious. There has been a practical upper limit to the masses of ions for which tandem mass spectra can be obtained with a hybrid,'-"Jwhich in the case of peptides lies around 800-1000 for a singly charged ion. It has been suggested that a major contributing factor to the problem of fragmenting large ions is collision energy (in the center-of-mass frame) being too small.ll For a stationary collision gas mgand a parent ion mi with energy E prior to collision, the center-of-mass collision energy ECOM is given by
Tandem mass spectrometry (MS/MS) offers advantages over single-stage mass spectrometry (MS) in terms of selectivity of analysis and specificity in molecular structure determination.'-3 The first mass analyzer (MS1) selectively transmits a particular m/z ratio, and following fragmentation the second analyzer (MS2) provides the mass spectrum of the selected ion. The benefits of tandem mass spectrometry for chemical analysis are becoming much more widely available due to advances in instrumentation. One particularly successful approach to tandem mass spectrometry has been to combine a double-focusingmass spectrometer of either forward (EB) or reverse (BE) geometry with a quadrupole mass analyzer (Q), producing a hybrid i n ~ t r u m e n t .It~ is ~ usually arranged so that fragmentation takes place within a radio frequency (r0-only quadrupole (q), located before the mass-analyzing quadrupole (Q).The advantages of hybrids arise with the high mass resolution available in MS1 for selection of parent ions, the unit mass-resolution of MS2 for analysis of fragment ions, and reduced complexity and cost compared to a four-sector tandem mass spectrometer.
Clearly, if the mass mi of the parent ion is much greater than the mass m, of the collision gas, that fraction (mg/(mi+ m,) of the ion's energy representing the collision energy ECOM becomes small. Bean et alS8 have concluded from a comparison of foursector and hybrid tandem mass spectra that for singly-charged peptides with M > 1OOO Da, more complete sequence information and lower limits of analysis are obtained from high-energyCAD on the four-sector instrument. The incident ion energies E employed on the four-sector were 5-10 keV, whilst those for collisions on the hybrid were much lower (10-20 eV). Alexander et aL7 making a similar comparison concluded that for peptide ions with M > 800Da the efficiency of CAD was low and highly dependent on the structure of the peptide when using the hybrid. The conclusions of these two recent studies reflect the consensus of opinion that hybrids are of limited usefulness for tandem mass spectrometry of
(1)McLafferty, F. W.,E d . T a n d e m M a s s S p e c t r o m e t r y ; Wiley-Interscience: New York, 1983. (2)Hunt, D. F.;Yates, J. R.; Shabanowitz, J.; Winston, S.; Hauer, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6233-6237. (3)Biemann, K.;Martin, S. A. Mass Spectrom. Reu. 1987, 6 , 1-76. (4) Schoen, A. E.; Amy, J. W.; Ciupek, J. D.; Cooks, R. G.; Dobberstein, P.; Jung, G. Int. J. Mass Spectrom, Ion Processes 1985, 65, 125-140. (5) McLuckey, S. A.; Glish, G. L.; Cooks, R. G. Int. J.Mass Spectrom. Ion Phys. 1981, 39, 219. (6)Glish, G. L.; McLuckey, S. A.; Ridley, T. Y.; Cooks, R. G. Int. J. Mass Spectrom. Ion Phys. 1982,41, 157.
(7)Alexander, A. J.;Thibault, P.; Boyd, R. K.; Curtis, J. M.; Rinehart, K. L. Int. J. Mass Spectrom. Ion Processes 1990, 98,107-134. (8)Bean, M. F.;Carr, S. A.; Thorne, G. C.; Reilly, M. H.; Gaskell, S. J. Anal. Chem. 1991,63, 1473-1481. (9)Poulter, L.; Taylor, L. C. E. Int. J. Mass Spectrom Ion Processes 1989. 91. 183-197. (10)Alexander, A. J.; Dyer, E. W.; Boyd, R. K. Rapid Commun. Mass Spectrom. 1989, 3, 10,364-372. (11)Neumann, G. M.; Sheil, M. M.; Derrick, P. J. Z . Naturforsch. 1984,39A, 584.
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0003-2700/92/0364-2628$03.00/0 0 1992 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
large ions (M > 1000 Da),7-9J2unless perhaps the ions are multiply charged as in the case of electrospray i0ni~ation.l~ It is generally agreed that the collision energy ECoM is too low for rapid fragmentation of a large ion to be induced, under the usual operating conditions for a hybrid whereby ions enter the rf-only quadrupole with energies of a few tens of electronvolts.10J2J4Consideration of expression 1for ECOM immediately points to one solution, which is to raise the incident ion energy E. Surface-induced dissociation, which in the terms of expression 1 means that mg a, has been exploredl5 but as yet has not been shown to be of practical utility for large ions. Alexander et 81.10 have discussed the use of higher ion energies ELM for CAD in rf-only quadrupoles and have pointed out that in order to achieve satisfactory transmissions an increase in ion energy ELMmust be accompanied by a rise in the value of the rf amplitude. E- is used to denote the energy gained by a singly charged ion on being accelerated from the source potential to the mean potential of the rf-only quadrupole. For the purpose of this discussion it is assumed that EM = E. Alexander et al.l0demonstrated convincingly that a portion of the tandem mass spectrum of a dynorphin fragment 1-9 could be clearly observed when ELM = 300 eV was employed at a zero-to-peakrf amplitude of 179V,whereas a t the same ELAB but with a lower rf amplitude of 60 V signal could not be clearly discerned above the noise. The effect of raising the rf amplitude is to increase the focusing action of the rf-only quadrupole and thereby increase the transmission of the high-mass precursor and fragment ions by raising the number of nodes n N O D to unity. n N O D is the number of times the coarse oscillations in the ion motion bring an ion back to the ion optical Following Schoen and Syka,l6 Alexander et al.10 give the following expression for n N O D
-
nNOD = LeV,/(2f.rr2r,2mi1JzELAB1J2)
L and ro are the length and inscribed radius, respectively, of an rf-only quadrupole. V,f and f are the amplitude and frequency, respectively, of the applied rf. Clearly, n N O D is proportional to Vrf and inversely proportional to mi1J2, according to this treatment. A potential drawback of increasing Vfi is that rf-only quadrupoles are high-pass mass filters,l'-'Q and in principle it would be possible to lose the low-massregion of a spectrum through using a very high value of Vrf. The modeling by Davis and Wright20 of ion trajectories for CAD in an rf-only quadrupole also predicted that efficient transmission of high-mass parent and fragment ions demanded very high values of Vd (perhaps kilovolts). These calculations showed that, given a sufficiently long rf-only quadrupole and sufficiently high values of Vfi, high-mass ions could be returned to the ion optical axis prior to mass analysis in the final quadrupole. In this paper, results are presented from CAD experiments using a hybrid instrument designed with the above consid(12)Bott, G.;Ogden, S.;Leary, J. A. Rapid Commum.Mass Spectrom. 1990,4,9, 341-344. (13)Smith, R.D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C.; Udseth, H. R. Anal. Chem. 1990,62,882-899. (14)Alexander, A. J.;Thibault, P.; Boyd, R. K. Rapid Commum.Mass Spectrom. 1989,3,2,30-34. (15)Wright,B.;Davis,S. C.;Tomer, K.B.;McGown,S. R.Proceedings of the 39th Annual Conference in Mass Spectrometry and Allied Topics, Nashville; ASMS East Lansing, MI, 1991;p 504. (16)Schoen, A. E.; Syka, J. E. P. Proceedings of the 34th Annual Conference on Mass Spectrometry and Allied Topics, Cincinnati; ASMS: East Lansing, MI, 1986;p 722. (17)Dawson, P. H.,Ed. Quadrupole Mass Spectrometry and its Applications; Elsevier: Amsterdam, 1976. (18)Dawson, P.H.Adu. Electron. Electron Phys. 1980,53,153. (19)Dawson, P.H.Adu. Electron. Electron Phys., Suppl. 1980,13E, 173. (20)Davis, S.C.; Wright, B. Rapid Commun.Mass Spectrom. 1990, 4, 186-197.
2829
erations in mind. Peptide [M+ HI+ions with masses above loo0 Da have been considered, and in d l cases improved performance in terms of the efficiency of CAD and quality of the tandem mas8 spectrum have been obtained through use of high values of the rf amplitude V,f in the rf-only quadrupole. EXPERIMENTAL SECTION Anew rf-only quadrupolewas installedwithin a Kratos Concept IHQ hybrid mass spectrometer to give EBqQ geometry (q being the new rf-only quadrupole). The rf-only quadrupole collision cell was constructed from hyperbolic-section rods 200 mm in length, with an inscribed radius of approximately 4 mm. The cell was driven from an rf power supply capable of delivering lo00 V zero-to-peak at 1.2 MHz. The collision cell could be maintained at potentials up to 500 V below that of the ion source; i.e. EUB could be set as high as 500 eV. The mass-analyzing quadrupole Q was driven using a scanning function designed to maintain the same ion energy for all ion masses within this quadrupole. Argon was used as the collision gas in all experiments; the transmission was 50% in all CAD experiments. A t E = 500 eV,ECOM for dynorphin A fragment 1-9(M + H = 1137.7) with argon as the target is 17 eV, which can be compared with ECOMof 14 eV at E = 4000 eV and helium target as might be employed on four-sector instruments. The rf-only quadrupoleq was scanned in unison with the massanalyzing quadrupole,maintaininga constant value (about 0.35) for,the Mathieu parameter of the rf-only quadrupole. Thus V,f was decreased linearly, as the masses of the fragment ions transmitted and detected were scanned downward. The values of Vd employed were such that in the case of the dynorphin A m/z 1137.7 ion at EL- = 500 eV there were three nodes (nNOD = 3) according to calculations. The mass resolution of the double-focusing mass spectrometer was 1000in all experiments. A mass resolution of about 1.5 mass units using a 50% valley definition was achieved at all masses with the mass-analyzingquadrupole. Spectra were collected and processed as raw data at a scan speed such that the whole spectrum would be scanned in 10 s. Scans were summed to improve signalto-noise. Acquisition times were between 3 and 5 min, with the exception of the measurement on 500 pmol of dynorphin A fragment 1-9 where acquisition was for 1 2 min. A Kratos Concept IIHH four-sector mass spectrometer of ElBlEzBz was used to obtain the high-energy CID spectra. The mass resolution of both MS1 and MS2 was 1000, the scan speed was 30 s/decadeover a calibratedmass range of 1150-14 for 8-keV ions. The collision gas was helium, and a beam attenuation of 70% was used at 4-keV collision energy (i.e. the collision cell floated at 4 kV). At 500-eV collision energy (collision cell at 7500 V) the beam attenuation used was 50 % since problems associated with electrical discharges occurred at higher gas pressures. Peptide samples were obtained from Sigma Chemical Co. and used without further purification. On the hybrid instrument, samples were ionized using a standard Kratos kiloelectronvolt caesium ion (LSIMS) source. Except where stated, a single loading of 1-10 nmol of peptide was used for each spectum recorded. The matrix used was m-nitrobenzyl alcohol with 1% trifluoroaceticacid. In the four-sectorexperiments,an Ion Tech saddle-field atom gun using xenon was employed. The matrix used was a 1:l mixture of glycerol and thioglycerol.
RESULTS (a) Dynorphin A Fragment 1-9. The [M + HI+ ion of dynorphin A fragment 1-9 (YGGFLRRIR-OH) was chosen as a test case, because in a previous study on a hybrid instrument7 it was shown to give a poor yield of fragment ions. The poor fragmentation was attributed to the localization of charge on the highly basic arginine residues at the C terminus.9 Specifically, Alexander et al.7 reported that very little useful information could be obtained using a hybrid a t either E- = 30 eV or ELM = 100 eV even when multiple sample loadings are used. Figure 1shows the tandem mass spectra of [M + HI+mlz 1137.5 measured using the hybrid
2090
ANALYTICAL CHEMISTRY, VOL. 64. NO. 21, NOVEMBER 1, 1992 Ar/C ID
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instrument a t E- = 100, 400, and 500 eV both with and without argon collision gas. At ELAB= 100 eV, the introduction of argon to induce CAD has not greatly increased fragmentation beyond the unsatisfactory level present in the absence of collision gas (at ELAB= 100 eV). At EL- = 400 eV, the presence of the collision gas has enhanced fragmentation significantly. The fragment ion peaks observed in the absence of collision gas are much diminished in importance, relative to the peaks appearing in the spectra as a result of CAD. This behavior is reenforced by the spectra at ELAB= 500 eV, where the differences between the spectra with and without collision gas are yet more pronounced (Figure 1). The quality of the MSIMS spectra must be assessed both in terms of the number of fragments present and upon the structural information which can be obtained from their assignment. At 100 eV the dominant fragment ions are bz, y3", bg, b,, and bs + HzO, which mainly reflects the location of arginine residues. In addition some small neutral losses from [M + HI+ (Le. loss of 60, 42, and 15) are present. The only significant difference between the spectra at 100eV with and without collision gas is the increase in relative abundance of the ion at m/z808 and of the immonium ions. Overall the spectra at 100 eV are unsatisfactory, containing too few ions
for structural assignment and this situation is virtually unchanged by collisional activation. In striking contrast, at E m = 400 or 500 eV, the benefit of using higher collision energy and high V,f is clearly illustrated by the CAD spectra shown in Figure ld,f. These spectra contain sequence ions across the mass range which are not present in the corresponding unimolecular spectra (Figures lc,e) and hence must arise via collisional activation. Of particular significance is the appearance of ions at mlz 654 (w5) which is thought to be due to the elimination of the isopropyl radical from the Leu side chain from 25 + 1, and a t m/z737 (d7) due to @ cleavage of an Arg side chain from a7 + l.zlSuch side chain cleavagesz1are generally not seen in low-energy CAD spectra since a high-energydeposition on collisional activation is required for them to occur.10 In addition the immonium ions for the residues Leu andlor Ile, Phe, and Tyr are present in high abundance. The high-energy MS/MS spectra of the dynorphin A (1-9) [M + H]+ion recorded on a four-sector instrument using He and using Ar collision gases are shown in Figure 2. These are (21) Johnson, R.S.; Martin, S. A.; Biemann, K.; Stults, J. T.;Watson,
J. T.Anal. Chem. 1987,59, 2621-2625.
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similar to those published previously and, in terms of structural information, are superior to even the results at EL- = 500 eV on the hybrid. Thus, additional major ions are present a t mlz 243 (wz),342 (wg), 498 (~41,768(VS), etc., all of which are expected to be absent a t low collisipn energies. In addition, it should be remembered that tbii extra information obtained using a four-sector instrument, is in addition to other major benefits to analysis, i.e. higher mass resolution for fragment ions (in this experiment a mass resolution of lo00 a t 10% valley as compared to 1.5-2 amu a t 50% valley definition) and the resulting higher mass accuracy. However, when the hybrid and four-sector results for dynorphin A are compared using similar laboratory collision energies (E-), the hybrid results look more favorable. Figure 2c shows the four-sector MSIMS spectrum of dynorphin A recorded using a nominal collision energy of 500 eV and argon collision gas. This spectrum shows a lot of similarities to the hybrid spectrum. It is not expected to be identical to the hybrid spectrum for several reasons. First, the collision regions differ
NO. 21, NOVEMBER 1, 1992
2891
greatly in their length (10 mm for the four-sector, 200 mm for the hybrid) and ion optical properties, and hence the average number of collisionsand interaction times will differ, Second, the four-sector instrument is designed to operate a t kiloelectronvolt collision energies, and while there is no great loss in transmission with the collision cell floated to 7.5 kV (for an accelerating voltage of 8 kV), other problems do arise. For example, some of the major peaks seen in Figure 2c (labeled as A) are artefacts arising from transitions in the fourth field-free region, which become prominent at cell voltages close to the accelerating voltage, as predicted previously.zz Nevertheless, with a few exceptions the major peaks in both hybrid and four-sector spectra at EL- = 500 eV are similar (Leu and/or Ile, Phe, and Tyr immonium ions, bz, b3, w5, d7, as, bs + HzO, Tyr side chain). Thus, it can be concluded that if fragment ions can be contained by and transmitted through quadrupole collision cells with high efficiency, such as has been achieved here a t E- = 500 eV, then similar results to those obtained in four-sector instruments should be attainable. Furthermore if kiloelectronvolt collision energies can be realized on hybrid instruments, hybrid spectra should then resemble four-sector spectra more closely. (b) Gramicidin S. The unimolecular and CAD MS/MS spectra of the m/z1141.7 [M + HI+ion of gramicidin S (cyclic Val-Om-Leu-Phe-Pro)from the hybrid instrument are shown in Figure 3. The unimolecularspectra vary little with collision energy between EL- = 30 eV and ELAB= 400 eV and, furthermore, are similar to a spectrum published previously at ELAB= 30 eV using a different hybrid.7 In that reference7 it was suggestedthat the fragmentation behavior of gramicidin S is dominated by the precollision internal energy imparted by FAB ionization rather than by the energy deposited by collisional activation. That conclusion is consistent with the results of this study, as shown in Figure 3b, which indicate that a t EL- = 30 eV there is little difference between the unimolecular and CID spectra. However the benefits of the present rf-only quadrupole arrangement become apparent at EL- = 200 eV and EL^ = 400 eV (Figure 3d,f) where the low-mass fragment ions are seen in high abundances. Clearly, these fragment ions arise via collisional activation of the precursor ions, although in this case these low-massions are also present in the unimolecular spectra, but in very low abundance. The fragment ions present in the gramicidin S spectra have been assigned previ~usly;~ a- and b-series ions are formed following ring opening predominantly between either Leu and Phe or between Phe and Pro. (c)Valinomycin. A further example of the enhancement in fragmentation on raising EL- is shown in Figures 4 and 5 for the case of the [M + Na]+ ion of the cyclic depsipeptide valinomycin (the cyclic trimer (Val-Lac-Val-HyV)s)at m/z 1133.6. The relative intensities of the fragment ions in the unimolecular spectra are more or less invariant over a range of values of EL^ from 20 to 400 eV. At values of E- below 50 eV there is little enhancement of the unimolecular spectra on adding collision gas. Major unimolecular fragment ions are observed at m/z 763,963, and 1062. The ion at mlz 763 is simply explained as being [(HyV-Val-Lac-Val)z+ Nal+, but the ions a t mlz 963 and 1062 appear one mass unit higher than the corresponding ions in the four-sector spectrum at EL^ = 4 keV,23 which were assigned as being [(HyV-ValLac-Val)z - HyV - Val + Na]+ and [(HyV-Val-Lac-Val)zVal - HyV - Val + Nal+, respectively. As EL- is raised from 150to 250 eV (Figure 4f,h), fragment ions due to CAD processes become relatively more intense (22) Boyd, R. K.; Harvan, D. J.;Hass,J. R. Int. J.Mass Spectrom. Ion Processes 1985,65, 273-286. (23) Curtis,J. M.;Bradley, C. D.; Derrick,P. J.;Sheil,M. M. Og. Mass Spectrom. 1992, 27, 502-507.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
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than unimolecular processes, which is especially evident at the low-mass end of the spectrum. For example,the fragment ions at mlz 222,294,and 393 having the structures [HyV Val + Na]+, [HyV - Val - Lac + Nal+, and [HyV-Val-LacVal) Na]+, respectively, are very weak in the unimolecular spectra, but among the most intense ions in the CAD spectra at these collision energies. At EL- = 400 eV the CAD spectrum (Figures4jand 5)startsto resemble the high-energy four-sector spectrum and exhibits quite extensive fragmentation which is probably a result of a greater degree of charge remote fragmentation,as suggestedpreviously.23 Presumably, such processes require a greater deposition of energy on collisional activation.’* Hence, some different fragment ions become relatively more intense, e.g. the ion at mlz 949 is assignedas [(HyV-Val-Lac-Val)z-HyV-Val- 0 + Naminus 2H]+; a t EL- = 4 keV this is the most intense fragment ion and so it would be expected to become relatively more intense if EL- could be raised further on the hybrid. Similarly, the fragment ion at mlz 779,assigned as [(HyV-Val-Lac-Val)z+ O]+, is the most intense fragment ion in the range m/z 300900 in the four-sector spectrum at EL- = 4 keV but only becomes a significant ion in the hybrid MSIMS spectra once E m is raised to 400 eV. It is clear from the above discussion that both the degree of fragmentation and the nature of the
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dissociation channels changes with EL-. Hence, it would be desirable to construct hybrid instruments with the ability to study CAD processes over a wide window of EL- values (ideally from a few electronvolts up to a few kiloelectronvolts) whilst high transmission is maintained acrossthe mass range. (d) Higher Mass Peptides. The preceding examples illustrate the application of laboratory collision energies of around a few hundred electronvolts in a hybrid instrument to molecularspecies of around mlz 1100-1200. It is of interest to see the effect of increasing the mass of the precursor ion and, hence, decreasing ECOM.As a limiting case an attempt was made to obtain an MSIMS spectrum of the mlz 2531 [M HI+ ion of the insulin A-chain (not shown). At this mass, ECOMis about half of that for the preceding examples. In addition the precursor ion intensity is considerably lower (even without attempting to resolve the isotope peaks by increasing MSI resolution), and equally important, the number of possible fragmentation pathways is greatly increased. It was found that both the unimolecular spectrum and the CAD MSIMS spectrum having EL- less than 300 eV contained very little fragmentation. However, as E m approached 500 eV, a series of peaks, some of which may correspond to y” ions, was observed. The poor signal-to-noise ratios mean that such a spectrum would not be useful in the case of an unknown.
+
ANALYTICAL CHEMISTRY, VOL. 64,
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ANALYTICAL CHEMISTRY, VOL. 64. NO. 21, NOVEMBER 1, 1992 *I.
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Flgure 5. Expanded version of Flgure 4j: valinomycin [M + Na]+ tandem mass spectrum using the hybrid at Eue = 400 eV with Ar colilslon gas. X = [ValLacValHyV] where Val, Lac, and HyV are vallne, lactic acid, and hydroxyisovalerlc acid residues, respectlvely.
Nonetheless, it can be concluded that for an ion of mlz 2500, center-of-mass collision energies high enough to induce fragmentation do begin to be accessed in the present instrument as ELAB approaches 500 eV.
DISCUSSION The tandem mass spectra (Figures 1-5) obtained with the hybrid establish very clearly that increasing the ion energy ELAB by 1order of magnitude (from tens of electronvolta to hundreds of electronvolts) has a profound effect on the degrees of fragmentation of peptide ions lying in the mlz range 1OOO2500. To be able to exploit the enhanced fragmentation necessitates the use of higher values of the rf amplitude V,f in the rf-only quadrupole, as proposed by Alexander et al.10 The degree of fragmentation of the peptides investigated at EM = 500 eV approaches that observed using kiloelectronvolt incident ion energies on four-sector instruments, but the fragmentation patterns for a given peptide ion are not identical in the two cases (hybrid and four-sector). Equation 1indicates that the center-of-mass collision energy for a given peptide can be made to be the same in a four-sector instrument as it is in a hybrid instrument with E- = 400 eV and Ar collision gas, in two ways. The first method is to reduce E m from the normal 4 keV down to the hybrid value of E m = 400 eV by increasing the float-voltage on the collision cell to 7.6 keV (for an accelerating voltage of 8 kV). In this way the collision proceeds with the projectile ion at the same velocity in either instrument, although the collision region is much longer in the rf-only quadrupole. The second method is to reduce the atomic mass of the target gas from 40 amu (Ar) down to 4 amu (He) whilst ELAB is kept at 4 keV. In this way, ECOM remains the same but since the projectile ion has 10 times more translational energy, its velocity is greater, and hence the interaction time is 0.3 (= 11410)times less, if interaction between projectile and target ion occurs over the same distance. Experimentally, it appears that when the collision occurs a t the same ECOMand ELABin both instruments, a similar fragmentation pattern is observed, once ion-optical and other experimental factors are taken into account (see Figures 1 and 2c). On the other hand with ECOM kept the same but ELAB = 4 keV for the four-sector and ELM = 400-500 eV for the hybrid, significantly more fragmentation including more side chain cleavages and charge remote fragmentation (Le. processes requiring a higher uptake of internal energy) are observed (see Figure 2a) with the four-sector. Thus, if it can be assumed that the fragment ion patterns observed truly reflect the fragmentation processes occurring within the collision cells (and are not for example highly distorted by mass discrimination effecta due to the quadrupole collision cell), then it would appear that ECOMis not the sole arbiter
of collisionallyactivated dissociation processes. One possible explanation is that at least some of the fragmentation reactions are highly sensitive to the interaction time t,. It has been suggested in an earlier study by Alexander et al.7 that the crucial quantity may be the so-calledMassey parameter R,Z49% (the ratio of tJ r , where T is the characteristic period of the internal mode which is being excited), rather than ECOM. When RM 1, a high probability for energy transfer occurs. For projectile ions of mlz 1100,R, = 1for translational-vibrational energy exchange over the range of ELAB from tens of electronvolts to kiloelectronvolts. The predicted energy transfer would be greater at kiloelectronvolt energies (He case on four-sector) than at hundreds of electronvolts (Ar case on hybrid); however the predicted energy uptake would not exceed about 1eV in either case. Other mechanisms for translational-to-internal energy exchange may occur predominantly at values of ELABin the kiloelectronvolt range (e.g. translational-electronic). Thus the intermediate values of ELABused in the hybrid described in this work (200-500 eV) may be at the threshold for fragmentation processes resulting from such excitation to occur. This idea is supported by the results presented here which show thresholds of incident ion energy for the formation of certain fragment ions, e.g. the ions at mlz 949 from the [M + Na]+ ion of valinomycin which has an onset in the hybrid spectra at between ELAB= 250 eV and E m = 400 eV and is the most intense fragment ion in the four-sector spectrum at ELM= 4 keV. A different viewpoint is afforded by a so-called “impulsive collision theory” (ICT) described recently.26 ICT is a simple mathematical model for impulsive energy transfer between a fast macromolecular ion and a stationary target gas atom. In ICT, it is assumed that energy and momentum transfer takes place between the target atom and a single atom (ma) of the projectile ions. It is implicitly supposed that the interaction time is short compared to the time required for energy redistribution within the ion, i.e. independent of rC. A logical result of this model is that the efficiency by which the center-of-mass collision energy ECOM is transferred into internal energy ( 8 )(the average internal energy uptake) is a t a maximum when the mass of the target atom mg closely matches the mass of participating atom from within the projectile ion. The efficiency factor X for transfer of the available center-of-mass collision energy into internal energy becomes unity when ma = mg, as given by the following simplified expression:26
-
(Q)= ’12XECOM where
X = 4mam,/(m, + ma)’
(3)
Equation 3 predicts that, in the present example for dynorphin A (1-91, if the assumption is made that all atoms in the macromolecular ion are equally likely to be involved in the collision, then (Q)= 0.34&0~for helium collision gas and ( 8 )= 0.19Eco~for argon collision gas. Thus, for a fixed ECOM,there is about twice as much internal energy uptake for collisions with helium as there is for collisions with argon. Such a significant difference in ( 8 )(about 2 eV in this case) might well explain the result described above, i.e. that with ECOM invariant, but using He collision gas for four-sector (at ELAB = 4 keV) and Ar collision gas for the hybrid ( E m = 400 eV), significantly more fragmentations including more side chain cleavagesand charge remote fragmentation are observed with the four-sector. ~~
(24) Massey, H. S. W. Rep. h o g . Phys. 1949,12, 248. (25) Beynon, J. H.; Boyd, R. K.; Brenton, A. G. Ado. Mass Spectrom. 1986,10,437-469 and references cited therein. (26) Uggerud, E.; Derrick, P. J. J . Phys. Chem. 1991,95, 1430-1436.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
CONCLUSION The useful mass range of magnetic sectorlquadrupole hybrid tandem mass spectrometers can be extended to mlz 2500, through using ion energies ( E m ) about 1 order of magnitude greater than has previously been usual (500 eV compared to 50 eV). This is made possible by raising the amplitude Vd of the rf potentials applied to the rf-only quadrupole. Higher values of Vfi can increase the focusing action of the rf-only quadrupole, so that high-mass ions with high energies are transmitted with high efficiencies. The success of this strategy is exemplifiedby the MSIMS spectrum of dynorphin A fragment 1-9. At EL- = 500 eV an MSIMS spectrum was obtained with 500 pmol of sample which gives satisfactory sequence information, whereas this had not been possible in a previous study.' This spectrum showed similar fragmentation to a spectrum obtained on the four-sector instrument with the same E m and collision gas. An additional finding was that when ECOM remained invariant, changingcollisiongasfromArtoHe (with therequiredchange in E M ) resulted in more high-energy fragmentation pathways
2635
(e.g. side chain cleavages). This implies a neater uDtake of internal energy with He collision gas in agreement with ICT,26 although the difference in available interaction time may also result in a shift in available mechanisms of translationalinternal energy exchange. Finally, the results point to the fact that it is desirable to construct hybrids capable of laboratory collision energies in the kiloelectronvolt range in order to achieve the most useful CID spectra. Work in this direction is currently in progress. ACKNOWLEDGMENT We are pleased to acknowledge financial support under the Teaching Company Scheme (SERC/DTI). C.D.B. thanks Kratos and SERC for a CASE Studentship. We are grateful to Margaret Hill for skillful assistance in the preparations of the manuscript. RECEIVED for review April 6, 1992. Accepted July 7,1992.
