Accelerated Articles Anal. Chem. 1994, 66, 2801-2808
Collisional Activation of Large Multiply Charged Ions Using Fourier Transform Mass Spectrometry Mlchael W. Senko, J. Paul Speir, and Fred W. McLafferty' Baker Laboratory, Department of Chemistry, Cornell University, Ithaca, New York 14853-130 1
For small singly charged ions, Fourier transform mass spectrometry ( m M S ) has demonstrated the ability to perform multistage mass spectral experiments (MS") with high resolution and high mass accuracy using collisionally activated dissociation (CAD). The combination of electrospray ionization (ESI) with the FTMS provides the potential to extend these capabilities for structural characterization of large biomolecules. The standard FTMS-CAD method is problematic in that it is inefficient and produces ions well away from the center of the cell. More efficient collisional methods have been demonstrated for small molecules (sustained off-resonance irradiation (SORI) CAD, very low energy (VLE) CAD, multiple excitation collisional activation (MECA)) that provide the additional benefit of producing product ions closer to the center of the trapped ion cell. The efficiency (>92%) for producing sequence-informative peaks from large multiply charged ions is far better than standard CAD. Disadvantages, such as blind spots where no product ions are observed, and isotopic distortions which can cause an incorrect mass assignment, must be considered when methods designed for small singly charged ions are applied to large multiply charged ions. SORI provided the highest efficiency, selectivity, and resolving power ( 9 X lo5) for product ion spectra and is recommended for most applications because of its ease of implementation. New techniques such as electrospray ionization (ESI)lv2 and matrix-assisted laser desorption/ionization (MALDI)3 have made possible accurate molecular weight determinations for large biological molecules. ESI has the particular benefit that multiple charging shifts the signal of molecules as large as 200 kDa down to the m / z range accessible with most mass a n a l y ~ e r s .Tandem ~ mass spectrometry (MS/MS) dissocia-
tion of such molecular ions can provide structural characterization, but collisionally activated dissociation (CAD) of singly charged ions has an upper limit of -3 kDa. With the multiple charging provided by ESI, this mass limit appears to be removed, with CAD spectra reported for 150-kDa ions.5 For large ions ( > l o kDa), dissociation has been performed in the high-pressure region of the source between the capillary and skimmer (nozzle/skimmer dissociation)6 and in the collision cell of a triple q ~ a d r u p o l e . ~ Multiple charging's benefit of providing ions amenable to dissociation is somewhat offset by the ambiguity of the charge state ( z ) and, therefore, mass (m).Typically, two peaks of different z but thesame m can beused to solve this ambiguity: but this method is difficult to apply to mixtures. Deconvolution algorithms have been developed to assist in these cases9JO but have yet to prove applicable to dissociation spectra. For true MS/MS where one charge state is selected and dissociated, typically fewer z values appear for each m as compared to nozzle/skimmer dissociation. As an example, the MS/MS spectrum of the (M + 19H)19+ of PA chain of hemoglobin produced 13 peaks whose m / z values correspond to fragments expected from the known sequence, but for only one of these could the m value be assigned from multiple z values." However, multiple charge states are not necessary if the resolving power is greater than the m value, as the resolved isotopic peaks provide an internal mass scale spaced 1 Da apart.12 The Fourier transform mass spectrometer (FT-
( I ) Fenn, J. B.; Mann, M.; Meng,C. K.; Wong, S. F.; Whithouse, C. M.Science
(4) Feng. R.; Konishi, Y . Anal. Chem. 1992, 64, 209C-5. (5) Feng, R.; Konishi, Y.Anal. Chem. 1993, 65, 645-9. (6) Loo, J. A.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1988, 2, 207-10. (7) Loo, J. A,; Edmonds, C. G.; Smith, R. D. Science 1990, 248, 2 0 1 4 . (8) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989,61, 1702-8. (9) Reinhold, B. B.; Reinhold, V. N. J . Am. Soc. Mass Specrrom. 1992,3,20715. (10) Labowsky, M.; Whitehouse, C.; Fenn, J. B. Rapid Commun. Mass Specrrom.
1989, 246, 64-7 1. (2) Loo, J. A.; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1990, 59, 158. (3) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B . T.Anal. Chem. 1991.63, 1193A-202A.
(11) Light-Wahl, K. J.; Loo, J. A.; Edmonds,C. G.;Smith, R. D.; Witkowska, H. E.; Shackleton, C. H. L.; Wu, C. C . Eiol. Mass Specrrom. 1993,22, 112-20. (12) Henry, K. D.; McLafferty, F. W. Org. Mass Specrrom. 1990, 25, 490-2.
0003-2700/94/0366-280 1$04.50/0 0 1994 American Chemical Society
1993, 7 , 71-84.
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MS)I33l4is perhaps the ideal mass analyzer for use with ESI because of its unmatched ability to obtain high mass accuracy, high sensitivity, and ultrahigh resolving powers simultaneously over a broad m / z range. This has been extensively demonstrated with FTMS, such as for nozzle/skimmer dissociation of ubiquitin (8.6 kDa)I5 and carbonic anhydrase (29 kDa), wheremasses could be assigned to over 100 isotopicclusters.16 Although nozzle/skimmer dissociation is simple to implement and can provide valuable structural information, it has two inherent weaknesses. First, it is non-mass-selective, so that it has limited applicability to mixtures unless timeconsuming chromatographic separation is performed beforehand. Second, it allows for only one stage of dissociation because the ions pass through the source only once. More extensive dissociation can be induced with greater voltage differences, but this is not as beneficial as multiple stages of mass spectrometry, because the source of the products is ambiguous. For FTMS, dissociation induced in the trapped ion cell holds more promise in that it is mass selective and can exploit the ability of the FTMS to perform MSn without the necessity of additional instrumentation. For collisional activation with FTMS, translational energy is typically provided to the precursor by resonant excitation of its cyclotron motion. Activation then occurs by collision with either a static17 or pulsedI8 background gas. Since activation and measurement are not separated spatially, but temporally, the pulsed gas method is preferable because it allows the system to return to a low-pressure state, which is optimal for high resolution detection. This resonant activation method has some inherent deficiencies. First, the amount of translational energy that can be given to the precursor is limited by the magnetic field and the size of the ion trap. Second, product ions are formed off axis (nonzero magnitron radius), limiting the number of further stages of M S and the radius of excitation that can beused for detection, which compromises signal and resolving power.I9 Third, ions are activated with a broad range of internal energies (glancing vs head-on collisions, various number of collisions),20 so that highly efficient precursor dissociations cannot be achieved without extensive secondary dissociation of the product ions. More efficient dissociation can be achieved while simultaneously producing product ions closer to the center of the cell by use of multiple excitation collisional activation (MECA).21 After low-level resonant excitation, undissociated precursor ions will tend to return to the center of the cell due to “frictional” collisions with the background gas, a method exploited for remeasuring the same ion population numerous times to improve signal-to-noise ratios.22 Precursor ions that do not dissociate after the first excitation can be re-excited (13) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, 215A-29A. (14) Koster,C.; Kahr, M . S.;Castoro, J. A.; Wilkins, C. L. MassSpectrom. Reo. 1992, I / , 495-512. ( 1 5 ) Loo, J. A.;Quinn,J . P.; Ryu,S. 1.; Henry, K. D.;Senko, M. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 286-9. (16) Senko. M. W.; Beu, S.C.; McLafferty, F. W. Anal. Chem. 1994,66, 415-7. (17) Cody, R. B.; Burnier, R. C.; Freiser, B. S. Anal. Chem. 1982, 54, 96-101. (18) Carlin, T. J.; Freiser, B. S. Anal. Chem. 1983, 55, 5 7 1 4 . (19) Guan, S.; Marshall, A. G.; Wahl, M . C. Anal. Chem. 1994, 66, 1363-7. (20) Burnier, R. C.; Cody, R. B.; Freiser, B. S . J . Am. Chem. Soc. 1982, 104, 743641, (21) Lee. S. A.; Jiao, C. Q.; Huang, Y.;Freiser, B. S . Rapid Commun. Mass Spectrom. 1993, 7, 819-21. (22) Williams, E. R.; Henry, K. D.; McLafferty, F. W. J . Am. Chem. Soc. 1990, 112, 6157-62.
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multiple times, without reexciting product ions, until dissociation occurs, improving efficiency (>50%) over standard CAD. The multiple excitations allow for significantly lower activation energies while high efficiency is still maintained, which will leave product ions closer to the center of the cell. In the initial report, naphthalene, an ion difficult to fragment by traditional CAD, produced abundant fragment ions using MECA.21 Another method for extended excitation utilizes the fact that ions excited slightly off-resonance will be alternately accelerated and decelerated with a period equal to the difference between the excitation frequency and the ion cyclotron frequency.23 A more complete evaluation of this “sustained off-resonance irradiation” (SORI) te~hnique2~ shows that it provides a more uniform activation than standard CAD. The slow activation from multiple low-energy collisions allows dissociation to occur near threshold, producing products only by the lowest energy pathways, such as the dissociation of protonated ethyl acetate with >90% efficiency to yield only one product.24 A similar method utilizes the previously demonstrated ability to deexcite previously excited ions by the use of a 180° phase shift of the excitation waveform.25 Using a resonant excitation and repeated phase shifts, ions are accelerated and decelerated numerous times to activate slowly the precursor through numerous low-energy collision^.^^^^^ The average kinetic energy of the precursor ion can be manipulated by this very-low-energy (VLE) collisional activation, which allows fine control of the average kinetic energy through the excitation voltage and the number of excitation cycles between phase inversions. Even without optimization, tert-butoxide anions were dissociated with high selectivity and 61 % effi~iency.~’ These three methods (MECA, SORI-CAD, VLE-CAD) all possess desirable features for collisional activation of large multiply charged ions and, therefore, have been chosen for a comparison of suitability for MSn structural studies of biological molecules. Yet another alternative method, photodissociation, is discussed in the accompanying papers2*
EXPERIMENTAL SECTION All proteins were obtained from Sigma (St. Louis, MO) and used without further purifications, except thioredoxin, which was obtained from Calbiochem (San Diego, CA). Solutions were electrosprayed at a concentration of 20 p M in 76% MeOH, 24% H20, and 2% HOAc. In the ESI-FTMS i n ~ t r u m e n t a t i o nions , ~ ~formed , ~ ~ at atmospheric pressure were guided through five stages of differential pumping using radio frequency-only quadrupoles to the trapped ion cell of a modified Extrel FTMS (Madison, WI) mass spectrometer (23) Heck, A. J . R.; de Koning, L. J.; Pinkse, F. A.; Nibbering, N. M. M . Rapid Commun. Muss Spectrom. 1991, 406-14. (24) Gauthier, J . W.; Trautman, T. R.; Jacobson, D.B. Ana/. Chim. Acta 1991, 246, 211-25. (25) Marshall, A. G.; Wang, T. L.; Ricca, T. L. Chem. Phys. Lett. 1984,105,233. ( 2 6 ) Boering, K. A.; Rolfe, J.; Brauman, J. I. Rapid Commun. Muss Sperrrom. 1992. 6. 303-5. (27) Boering; K. A.;Rolfe, J.; Brauman, J. I. Int. J . MassSpectrom. IonProcesses 1992. ~. 117.. 357-86. (28) Little, D. P.;Speir, J. P.; Senko, M. W.; McLafferty, F. W. Anal. Chem., following paper in the issue. (29) Beu, S . C.;Senko, M. W.; Quinn, J . P.; McLafferty, F. W. J . Am. Soc. Mass Specrrom. 1993, 4, 19&2. (30) Beu, S.C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty, F. W. J . Am. Soc. Mass Spectrom. 1993, 4, 557-65. ~~
~
equipped with a 6-T superconducting magnet and an Odyssey data system. Pulsed argon or nitrogen was used to assist trapping and as a collision gas at an indicated pressure between 10" and 10" Torr; true pulsed pressures at the cell may be 1 order of magnitude higher. The entire isotopic envelope of ions for the desired precursor was isolated using stored waveform inverse Fourier transform (SWIFT) e ~ c i t a t i o n . ~ ~ Isolation of a single isotopic peak of the precursor would significantly narrow the isotopic distributions of the product ions and thus make difficult the assignment of charge and the proper monoisotopic mass. Optimal excitation voltage for activation was determined by first overattenuating, so no dissociation was observed, and then gradually decreasing the attenuation until the precursor was reduced to -1% of its initial abundance. For data acquisition, chirp excitation from 50 to 150 kHz was performed at a sweep rate of 150 Hz/ps before detection of a single scan of 256K data points in direct mode at an acquisition rate of 400 kHz, except where noted. Standard CAD was performed by pulsing in the collision gas, and waiting 0.25 s to allow the background pressure to increase, before resonant excitation of the precursor to 165 eV. For MECA, typically ions were excited to 25 eV and then allowed to relax for 5 ms. This process was repeated 400 times before spectral acquisition. This lower energy, higher repetition method was found to yield more reproducible results than the parameters used in the original report.*I SORI was performed 2-kHz off-resonance for 1 or 2 s, with an average kinetic energy of 14 eV. In the original adjustment of the difference frequency was used for fine control of the kinetic energy, since the data system limited attenuation of the excitation voltage to whole decibels; here, the Odyssey data system allows fine control of the excitation voltage and thus of the average kinetic energy, so adjustment of the difference frequency was unnecessary. In contrast to the original analog implementation of VLE-CAD,26 this work uses a digital implementation, where VLE waveforms are created in PV-Wave (a visual data analysis package, Visual Numerics, Houston, TX), and stored as SWIFT waveforms, to be downloaded to the instrument by the Odyssey data system. Typically, the precursor's cyclotron motion was excited for 25 cycles before phase inversion, for an average kinetic energy of 10 eV. The effective dissociation efficiency was calculated by summing the abundance of all isotopic peaks of product ions with greater than 3:l signal-to-noise ratio (excluding M xH2O peaks) and dividing by the precursor ion abundance obtained without activation. Loss of H 2 0 from the precursor is not considered an informative dissociation, so is excluded from the efficiency calculations. Product ions will always have a narrower isotopic distribution than their precursors, so that counting only the most abundant peak will cause a significant overestimation of the efficiency. RESULTS AND DISCUSSION Bovine ubiquitin (8.6 kDa) was used for comparison of the various methods because its nozzle/skimmer and MS/MS ~~
~
(31) MarshalLA. G.; Wang, T.-C. L.; Ricca, T. L. J . Am. Chem.Soc. 1985,107, 1893-7.
i
V I
I
til+
I
111
I
spectra from quadrupole32and FTMS instrument^'^ have been published previously. Using established nomenclature, cleavage of an amide bond produces y-type ions retaining the C-terminus and b-type ions retaining the N - t e r m i n u ~ .The ~~ predominant product in all cases is the y58, which is due to cleavage between a glutamic acid at position 18 and a proline at position 19. Favored fragmentation on the N-terminal side of prolines,16J2 particularly prolines with adjacent acidic residues,34has been reported previouslyfor peptide and protein cations. Using the FTMS, standard CAD of the (M + 1 lH)"+ precursor (165 eV, argon collision gas, 1 X 1P5Torr) produces a product ion spectrum very similar to the quadrupole MS/ MS spectra, with y 5 P as the predominant product. Minor products include the b1s2+,b52'+, ~ 2 4 ~(b~ry58)34~+, +, and others as labeled (Figure la). The base peak is undissociated molecular ion (abundance 27% of that of precursor without activation), and efficiency is less than 30% obviously limiting the number of stages n of MSn that can be performed. Nonconventional CAD. The three nonconventional CAD methods produced dramatically more efficient dissociation, even with pulsed gas pressures far lower ( 1 P Torr) than the standard method. (Figure 1 b-d). SORI-CAD proved most efficient at 92% followed by resonant amplitude modulated (RAM)-CAD (modified VLE-CAD; seediscussion below) at 89% and MECA at 80%. The abundance of undissociated molecular ion and its associated water loss accounts for -5% of the total signal, so that almost no loss of ions occurs using SORI. SORI and RAM-CAD produced remarkably similar product ion spectra (Figure 1b,c), con-
-
(32) LOO,J. A.; Edmonds, C. G.; Smith, R. D.Anal. Chem. 1993, 65,425-38. (33) Roepstorff, P.; Fohlman, J. Blomed. Mass Specrrom. 1984, J I , 601. (34) Yu,W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 301 5-23.
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2803
5 Y3?
.
8+
YE8
I
e+ Yso,
'
-H,O bm7+
10 10
y69i+
..
_I
b5I6+
I
mlz
300
150
100
+
Figure2. High-resolution SORI spectrumof (M 10H)l0+from bovine ubiquitin collected in heterodyne mode with 2M data points. Inset shows b# product ion wlth resolving power of >9 X lo5. time (sec)
sistent with the similar motion of the precursor during the activation process. The original SORI and VLE-CAD r e p o r t ~ described ~ ~ , ~ ~ the ability to vary the selectivity by altering the pressure and atomic/molecular weight (Le., Ar vs He) of the activation gas. A small increase in selectivity was observed for multiply charged ions on changing the pulsed gas pressure from 1 X 10-6 to 1 X 1e5 Torr, but this is primarily attributed to larger magnetron radii (and thus lower abundances) for lower molecular weight products caused by the higher gas pressures, and not increased dissociation selectivity. The lower efficiency of MECA may be explained by its higher energy activation, which produces far more secondary products (approximately twice as many total products as SORI), with signal-to-noise levels inadequate for inclusion in the efficiency calculations. A prime example is the (b52y5~)34~+ ion at m / z 952 (also from standard CAD, Figure la), formed by two cleavages of the backbone. Both its potential precursors, b52 and y58, are sufficiently far in m / z from the precursor to preclude activation during subsequent excitations; sufficient excess energy is given to the precursor to induce multiple fragmentations. More extensive dissociation of the precursor, which could be desirable for targeted structure conformations, was effected with MECA using fewer excitations with higher voltages and longer delays between repetitions. Coincidentally, both SORI and RAM produced a product at m / z 952 (Figure 1 b,c). In these cases, this was not primarily the internal fragment, but the overlapping b172+. Products of the same m / z , but different mass, with superimposed isotopic envelopes can be distinguished because the isotopic peaks will not be superimposed, demonstrating an additional advantage of high resolution. Another benefit of all the above methods is the minimal distortion caused to the ion cloud. Standard CAD produced time domain spectra lasting less than 1 s which provide resolving powers of less than 100 000 for the products. For the (M + 10H)lO+of bovine ubiquitin, SORI produced time domain spectra lasting in excess of 10 s and resolving powers approaching 1 X lo6 for the b# ion by using a lower pulsed gas pressure, while consuming only 500 fmol of sample (Figure 2). Such resolution capabilities make sub-ppm mass accuracy possible; for example, this will distinguish 20-kDa ions that only differ by replacing lysine (residual mass 128.095 Da) with glutamine (128.059 Da).29 SORI of laser-desorbed ions has recently been demonstrated in combination with quadrupolar axialization of the product ions, which provides greater 2804
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+
Flgure 3. Appearance/depletion curve of SORI of (M 10H)lO+from bovine ubiquitin showing no reduction of efficiency when overextended activation times are used.
sensitivity and resolving power (20K) than available without a x i a l i z a t i ~ n . The ~ ~ higher resolving powers available here without axialization are most likely due to a smaller spatial distribution of precursor ions as well as larger products, which can maintain small magnetron radii at the high background pressures necessary for dissociation. Dissociation Trends. A major benefit of these low-energy activation methods is that they minimize secondary fragmentation while optimizing efficiency. Using a true collision cell (as with a quadrupole or sector instrument), or nozzle/ skimmer dissociation, product ions continue to be activated until they leave the collision cell, which can cause secondary fragmentation, producing internal ions and smaller terminal products that can complicate spectral interpretation. This is demonstrated in the nozzle/skimmer and MS/MS spectra of bovine ubiquitin recorded on a triple quadrupole in~trument.~2 If simple cleavages occurred, fragments should exist as complementary ion pairs. In the quadrupole MS/MS spectra of the (M 1OH)l0+,an abundant product was the y244+,yet no complementary b526+ was observed. This suggests that either the b52 has been further dissociated or that the ~ 2 was 4 formed by a secondary fragmentation of a larger y ion, most likely the y58. For tryptic peptides, it has recently been demonstrated that b-type ions are more susceptible than y-type ions to further d i s s ~ c i a t i o n ,and ~ ~ this may explain their conspicuous absence from previously published CAD spectra of larger molecules.32 In contrast to the quadrupole results, the base peak for the SORI spectrum of the 10+ molecular ion of ubiquitin is the b5z6+ (Figure 2). Fragmentation that changes the m / z value changes the cyclotron frequency, so that further activation of the product ceases immediately, although activation continues for the precursor. Although complete dissociation of the precursor is typically obtained in less than 1 s, extending the activation to 3 s or more causes no reduction in the abundances of the products (Figure 3). For an unknown this simplifies optimization since excessively long activation can be used to ensure high efficiencies. Single cleavages of the backbone provide spectra that are far simpler to interpret and present the possibility of performing more sophisticated mass spectral experiments. This would
+
(35) Tang, X.;Boyd, R. K. Rapid Commun. Mass Spectrom. 1992, 6, 651-7.
involve further dissociation of each fragment from a complementary product ion pair, producing a complementary ion pair for that fragment. This would be continued until the fragments produced werein the 1-2-kDa range, at which size complete sequence information can presently be obtained from one CAD experiment. MS4has been performed for molecules like ubiquitin, but further stages are not possible without the benefit of signal averaging or remeasurement,22 due to signalto-noise ratios limited by space charge restrictions on the initial ion population. The promise of this method for complete protein sequencing is indicated by the initial MS/MS results on the carbonic anhydrase.16 An important feature for the interpretation of unknown spectra is that fragmentation occurs in a predictable fashion. Dissociation using the nonstandard collisional activation methods follows trends previously observed on other types of instruments. For compounds studied to date (insulin, ubiquitin, thioredoxin, cytochrome c, myoglobin), fragmentation occurs primarily on the C-terminal side of glutamic and aspartic acid residues. Metastable fragmentation adjacent to acidic residues has been observed previously for MALDIgenerated ions on a reflection time-of-flight in~trument.~4 Lower abundance products observed from the N-terminal side of the acidic residues assists in identifying the terminus associated with each fragment (b51-b52 pair, y59-y58 pair; Figure 2).16 Dissociation is enhanced when the residue on the C-terminal side of the acidic residue is proline, as demonstrated by the abundant y58 of bovine ~ b i q u i t i n . For ~ ~ the larger compounds (cytochrome c, myoglobin), dissociation is also observed adjacent to basic residues and is particularly favored when lysine occurs on the N-terminal side of a histidine. Loo et al. have previously suggested that fragmentation produced by low-energy CAD may be influenced not only by primary structure but also by secondary structure that is maintained in the gas phase.32 SORI-CAD results from ubiquitin, thioredoxin, and cytochrome c corroborate Loo's proposal. For ubiquitin, seven segments are considered to contain turns based on the X-ray crystal structure.36 Four of these areas (residues 18-21, 37-40, 51-54, 55-60) show fragmentation at nearby acidic units. Of the three remaining turns, two do not possess nearby acidic residues (7-10, 4548), leaving only one turn in the molecule that contains a nearby acidic turn that did not show fragmentation (62-65). Although this correlation might be attributed to acidic units having a tendency to appear in turns, five additional acidic residues exist in the molecule (21,24, 32, 34,64) that do not occur at turns, of which only one (b32/y44) shows fragmentation. Solution conditions used in these experiments (76% MeOH, 24% HzO, 2% HOAc) have been described previously as "denaturing" for bovine ubiquitin on the basis of circular dichroism and D20 exchange,37so fragmentation observed at turns in the structure would suggest that the protein has regained a significant amount of secondary structure upon conversion to the gas phase. These correlations between lowenergy CAD fragmentation and secondary structure support previous gas phase D20 exchange experiment^^^ that dem~~~
(36) Vijay-Kumar, S.; Bugg, C. E.; Wilkinson, K. D.; Vierstra, R. D.; Hatfield, P. M.; Cook, W. J. J . Biol. Chem. 1987,262, 6396-9. (37) Katta, K.;Chait, B. T. J . Am. Chem. Soe. 1993,115, 6317-21. (38) Suckau, D.; Shi, Y.;Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1993,90, 79CL3.
b
/I 815
817 mlz
819
+
Figure 4. Spectra of the ysss+ion produced by MECA of (M 1 lH)'l+ from bovine ubiquitin using (a) 1 2 . 3 5 4 excitation with a 4 X lo4 8 delay between repetitionsand (b) 13.084 excitation with a 5 X 10-3 s delay.
onstrated that large multiply charged proteins maintain a significant amount of their secondary structure for extended periods of time in the absence of solvent. Detailed relationships between secondary structure and location of fragmentation are currently being investigated. Complications with Large Ions. The application of MS/ MS to large molecules necessitates certain precautions that can typically be disregarded for small molecules. For large multiply charged ions, the monoisotopic peak can be of an unobservable abundance, so that determination of unit molecular weight must be made by a comparison of the measured and predicted isotopic distribution of a similar molecular weight.3g Thus, any uneven activation of the precursor that causes distortion to the isotopic distribution of the products could lead to an error in molecular weight determination. MECA is a resonant process, so that the incorrect activation frequency or power can cause isotopic distortions. MECA of the (M + l l H ) l l + ion of bovine ubiquitin (8.6 kDa), using 12.35-V excitation with a 4-ms delay between each of 400 repetitions, efficiently produces the ySg8+ fragment with the expected isotopic distribution (Figure 4a). However, increasing the excitation to 13.08 V and the delay to 5 ms causes a severe distortion of the isotopic distribution (Figure 4b). This distortion may be due to incomplete cyclotron relaxation of the undissociated precursor, and the cause is being investigated further. A potential solution to the uneven activation is the use of a narrow mass range SWIFT31 in place of the single-frequency excitation. A nonresonant process such as SORI should cause the side of the isotopic envelope closest in frequency to the activation to have a higher average kinetic energy than the side further from the activation frequency. The use of a large difference frequency will minimize isotopicdistortions but simultaneously causes another problem. For SORI, the off-resonance activation frequency will be resonant with a range of m l z values, which will cause any products formed in this m l z range to be either further activated or ejected from the cell entirely. For singly charged ions this is not a serious problem, because an activation frequency resonant with a higher m l z value than the precursor will not affect fragment ions. However, for multiply charged ions, products will appear on (39) Senko, M. W.; Beu, S. C.; McLafferty, F. W., submitted for publication in J . Am. SOC.Mass Spectrom.
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'a
I b
15 Y133
M-H,O
*
Illl
'
time
b
I1 L tiAe
1000 150i122000 2500
P+
10 0 1050 m z
950
Flgure5. SORI MS/MS spectra of (M 17H)17+from equine myoglobin with activation 2 kHz (a) above and (b) below resonance.
both the low- and high-mlz side of the precursor, creating a blind spot analogous to the 'black and "black canyonsn41seen for MS/MS spectra of quadrupole ion traps. This blind spot will be centered at the activation frequency and will be twice the width of the difference frequency. For SORI-CAD of the (M 17H)17+ from equine myoglobin, activation 2 kHz above the cyclotron frequency of the precursor produces abundant fragments between m/z 1000 and 1050, with no fragments observed between m l z 950 and 990 (Figure 5a). Activation 2 kHz below the precursor frequency produces fragments between m/z 950 and 1000 with no fragments between m / z 1000 and 1040 (Figure 5b). This blind spot necessitates two experiments to obtain a complete product ion spectrum for multiply charged ions using SORI. Empirically, 2 kHz has been determined to be a difference frequency that eliminates any isotopic distortion and minimizes the width of the blind spot. For a 6-T magnetic field and a difference frequency of 2 kHz, the blind spot is 40 m/z wide at m l z 1000. SORI at lower magnetic fieldsusing thesame activation energy will increase this m/z range.
+
Although VLE-CAD uses a resonant excite, it too is afflicted by blind spots. Due to the abrupt phase inversion, evenly spaced spikes are observed in the frequency domain spectrum of the activation waveform with decreasing amplitude away from the fundamental (Figure 6a). These spikes will cause further activation or ejection of product ions of corresponding cyclotron frequencies. For singly charged ions this problem is magnified by the fact that the spikes are closer together at masses less than the precursor due to the inverse relationship between frequency and mlz. To reduce the abruptness of the phase inversion, VLE-CAD waveforms were generated for this work by modulating the amplitude of the resonant frequency with a sine wave (RAM-CAD), as (40) Guidugli, F.; Traldi, P. Rapid Commun.Mass Spectrom. 1991, 5, 343-8. (41) Guidugli, R.; Traldi, P.; Franklin, A. M.; Langford, M. L.; Murrell, J.;Todd, J. F. J. Rapid Commun. Mass Spectrom. 1992, 6. 229-31.
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Flgure 6. Excitation of ion mlz values with VLE activation using (a) direct phase inversionand (b) sine wave amplitude modulation. Insets show activation waveforms. Horizontallines indicate threshold for ion ejection from the cell.
suggested by S . C. B ~ u The . ~ resulting ~ frequency domain spectrum has only two spikes, the sum and difference frequency of the activation and modulation signals (Figure 6b), and produces slightly more efficient dissociation than a true VLECAD waveform. Another difficulty encountered with multiply charged ions is the selection of an appropriate charge state for dissociation. SORI-CAD of the 9+ to 12+ charge states of bovine ubiquitin show that the different charge states produce dramatically different product ion spectra (Figure 7). Although the products observed are similar, the relative abundances and dissociation efficiencies vary dramatically. The poor quality of the 9+ and 12+ product ion spectra can be attributed partially to lower efficiencies, but a greater cause is the electrospray source's production of lower abundances of these charge states as compared to the 10+ and 11+. As previously discussed, the 11+ charge state produces an abundant y5S8+ and efficiency is >92%. However, the base peak from the dissociation of the 12+ charge state is the loss of water, and overall efficiency is
