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Tandem Fourier-transform mass spectrometry
85
International Journal of Mass Spectrometry and Zon Processes, 72 (1986) 85-91 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
TANDEM
FRED
FOURIER-TRANSFORM
W. McLAFFERTY
Chemistry Department, (First received
MASS SPECTROMETRY
** and I. JONATHAN
*
AMSTER
Cornell University, Ithaca, NY 148.53-1301 (U.S.A)
7 November
1985; in final form 30 June 1986)
ABSTRACT Advantages of the Fourier-transform mass spectrometer for tandem mass spectrometry (MS/MS) include the ability to perform consecutive mass separations (even MS”) in its one analyzer cell, unusually high resolution (lo6 at m/z 300) which is independent of ion kinetic energy, simultaneous (multichannel) detection of nearly all masses, sub-picomole sensitivity, high mass range ( >16000 daltons) without detector limitations, collisionally activated dissociation of ions of energies of volts to kilovolts with efficient daughter ion trapping, and flexible capabilites for ion/molecule reactions and ion photodissociation.
INTRODUCTION
The impressive recent advances in both Fourier-transform (FT) mass spectrometry and tandem mass spectrometry (MS/MS) make appropriate a review of their combined capabilities. MS/MS [l] can improve the specificity and sensitivity of MS with little sacrifice of its speed or accuracy. The first mass spectrometer (MS-I) can be used to separate molecular ions of mixture components produced by “soft” ionization, analogous to the chromatographic separation of GC/MS and LC/MS techniques. The separated molecular ion species can then be fragmented, for example by collisionally activated dissociation (CAD) or photodissociation. The resulting product ions separated in MS-II provide a secondary mass spectrum useful for structural characterization of the original molecular component [2,3] in much the same way that a normal mass spectrum is structurally characteristic of a pure sample. A second general use of MS/MS is to obtain additional structural information on pure compounds [4-71. “Hard” ionization, such as
Presented at the First European Symposium on Fourier-Transform Metz, France, 19 September 1985. ** To whom correspondence should be addressed. l
0168-1176/86/$03.50
0 1986 Elsevier Science Publishers
B.V.
Mass
Spectrometry,
86
that effected with 70 eV electrons, of a molecule produces fragment ions. Mass separation of these by MS-I and dissociation give MS-II mass spectra characteristic of the structures (even the stereochemistry [7]) of the individual molecular fragments, for which normal mass spectrometry with exact mass data only provides elemental composition assignments. However, most MS/MS instruments used to date have required a second mass analyzer, a substantial disadvantage in cost. In contrast, the ion cell of the FT mass spectrometer [8-121 can be used to select primary ions of a specific mass, dissociate or react these, and measure the secondary ions thus formed. Such an MS/MS capability with CAD was first demonstrated in 1968 [13] using the “double resonance” technique of ion cyclotron resonance spectrometry, the forerunner of FTMS. Although the unique capabilities of FTMS for normal mass spectrometry have been reviewed recently [9-121, both FTMS and MS/MS are progressing so rapidly that it appeared appropriate to review FTMS/FTMS and outline promising areas for future development . ION TRAPPING AND MEASUREMENT
FTMS employs “static” trapping of ions. Those moving at less than escape velocities orthogonal to the magnetic field (X and Y axes) are trapped in cyclotron orbits, while motions parallel to the field are contained by trapping plates with a small (e.g. 1 V) potential of the same charge. For mass measurement, all ions are accelerated with a broad-band rf frequency to orbits within the cell using the X-axis cell boundary plates. The Y-axis plates are then used to detect the orbiting packets of ions, each of which produces a complementary rf signal of magnitude proportional to the number of ions. Ions of many m/z values produce a corresponding multiplicity of overlapping frequencies; from these, the Fourier transform produces the mass spectrum, thus measuring all ions simultaneously. In contrast, scanning MS instruments must generate ions continuously, but at any instant can measure only those ions in a small mass range; to increase resolution, the width of this range must be decreased. Even for single-ion monitoring, the sensitivity of FTMS appears to be competitive with other MS instrumentation. For example, Gross and co-workers report [14] the detection of lo-i1 g of naphthalene at a resolution (m/Am) of 20 000 using multiphoton ionization with FTMS. MS/MS
For collisional dissociation (or ejection), ions of any chosen m/z value can be accelerated, providing an MS-I selection process. The secondary mass
87
spectrum can be measured by the same technique as that described for the primary ions, with the further advantage that the cyclotron frequency of an ion is only dependent on its mass-to-charge ratio (m/z) and the magnetic field, not on its kinetic energy. In contrast, magnetic and electrostatic analyzers often used as MS-II do not exhibit this kinetic energy independence and so the MS-II resolution is usually relatively poor ( < 100) because of the kinetic energy released in ion dissociation. Further, in such magnetic instruments with multi-kilovolt in acceleration, focusing of the secondary ions produced by CAD for introduction into MS-II is inefficient, with a collection angle of < l”, while FTMS ion trapping, especially in the X and Y axes, is quite efficient. For the low energy (lo-150 V) collisions in the triple quadrupole MS/MS instrument, the central rf-only quadrupole is similarly advantageous for focusing product ions scattered through larger angles [15]. CAD in magnetic instruments without special modification involves multi-kilovolt ion energies; with FTMS, such collision energies can be from the lowest values [16] up to the kilovolt range (2 keV for C,Hz’ ions in a 2.22 cm radius ion cell, 3T magnet [17]). Recent work by Russell [18] and Cody et al. [19] indicates that CAD of high mass (1000-2000 daltons) ions of sub-kilovolt energies may be much more effective than indicated by previous experiments with multi-kilovolt energies [20]. HIGH-RESOLUTION MS/MS
Tandem double-focusing mass spectrometers now available commercially can achieve resolving powers up to 100 000 in MS-I and 10 000 in MS-II (our homemade instrument, the first of this kind used in molecular studies [21], exhibits values of 50000 and 2000, respectively). FTMS, costing less than half the amount of these instruments, has achieved far higher resolution, such as 4 x lo6 at m/z 131 for MS-I [ll] and 330000 at m/z 145 in MS-II [22]. Such high FTMS resolution demands ion cell pressures near 10e9 torr, but these appear to be achievable conveniently by separate formation of ions outside the measurement cell; the dual cell developed by Nicolet Instruments appears to be particularly promising for this [ll]. Obviously, this resolving power makes possible a correspondingly impressive improvement in selectivity for MS/MS experiments. For example, consider the problem of the identification of the nerve gas sarin with high confidence. Of the 400 compounds of molecular weight 140 in our file of reference mass spectra, sarin is the only one which has the composition CqHi,,FOzP. Its mass differs by one part in 235000 from that of C,HsN,S, represented in the file by three compounds, and by l/141 000 from that of C,H,Cl, represented by 13 compounds (such as chloroxylenes). Thus, measurement of the mass of an
88
unknown m/z 140 ion with sufficient accuracy and resolution, in either MS-I or MS-II, depending on the experiment, would greatly increase the confidence in the identification of sarin, or prevent false alarms if the ions were actually of another composition. Selective rf excitation of primary ions at present can only be achieved with a resolving power of - 1000 [19], but the tailored excitation function developed by Marshall et al. [23] promises to give resolution in the selection process comparable with that achievable in the measurement process. FTMS resolution decreases linearly with increasing mass, so that 4 X lo6 resolution at m/z 100 predicts only unit resolution at m/z 20000. However, doublefocusing magnetic and time-of-flight instruments achieve only unit resolution or less above m/z - 5000.
MULTICHANNEL FTMS/FTMS
We recently suggested [24] another possible application of Marshall’s tailored excitation function to achieve the multichannel advantage simultaneously for MS-I and MS-II. At present, for the structural characterization of the multiplicity of fragment peaks in the spectrum of a large molecule, it is necessary to select and dissociate each primary ion individually, even though the measurement of the resulting MS-II spectrum can be done simultaneously with the multichannel advantage of FTMS. Thus, more sample must be ionized for each MS-II spectrum desired. If all primary ions are dissociated simultaneously, the resulting secondary spectrum shows the products of all primary ion dissociations together. To differentiate these, the CAD of all ions could be repeated, but using the tailored excitation function to dissociate differing proportions of the individual mass values. This could be done by reducing individual abundances through selective excitation to maximum orbits in which a fraction of the ions strike the cell walls. A Hadamard-like decoding function could then use the change in product ion abundances to assign precursor-daughter ion relationships. For example, if precursor ions of a particular m/z value are excited so that 50% are lost and all remaining ions are dissociated under the conditions of the original CAD spectrum, any daughter ions arising only from the selected m/z value should be reduced to 50% of their original abundance. The tendency of the most abundant CAD product ions to be formed by the loss of relatively small neutrals should reduce the degree of overlap in the simultaneous collisional dissociation of all fragment ions from a large molecule. Ambiguities remaining after one such modified CAD experiment could be clarified in a further experiment with appropriate abundance adjustments.
89 MASS RANGE
With rf circuitry giving sufficiently low frequencies, the mass range of FTMS should be virtually unlimited (> 100000) 191, although increasing the magnetic field increases both the resolution and sensitivity achievable at a ions at m/z 16241 have been detected by specific mass. Recently, Cs,& FTMS with good sensitivity [25]. The instrument used has a 3T magnet and this mass represents the limit of the rf excitation circuitry of the instrument. All masses from 100 to 16000 produced in a single ionization pulse were measured simultaneously. Dramatic progress has been made in the last few years (as described earlier [ 111) in several methods to ionize large molecules such as trypsin (molecular weight 23463) [26] by 252Cf plasma desorption [27]. In preliminary experiments using this method with FTMS, molecular ion species have been formed from leu-enkephalin [28] and gramicidin S [29]. As a recent example of FTMS/FTMS [19], laser ionization of - 10e8 g of the gramicidin D, a mixture of linear pentadecapeptides gave (M + K)+ ions of four components. Collisional dissociation of the m/z 1920-1921 ions gives a unit resolution CAD mass spectrum representing the major component. Differences corresponding to amino acid masses identified 11 residues from the N-terminal peaks and 8 from the C-terminal peaks, which with overlapping provided sequence information on 12 of the 15 amino acids of this component. Similar laser ionization of - lo-’ g of impure gramicidin S followed by CAD of its major (M + K)+ ion at m/z 1179 gave complete information on the amino acid sequence of this cyclic decapeptide. The resolution and mass accuracy make possible the clear distinction of the unusual amino acid ornithine, mass 114, from leucine, mass 113. OTHER METHODS
FOR CHARACTERIZING
PRIMARY
IONS
Besides both high- and low-energy CAD, other promising methods providing complementary structural information are made possible by the orders-of-magnitude longer ion residence times in FTMS. As pioneered using the forerunner technique of ion cyclotron resonance, these include ion/molecule reactions [13], electron bombardment [30], and laser photodissociation [31-331. Only our knowledge of ion/molecule chemistry limits the possibilities of the first, such as determining the H-D exchangeable hydrogen atoms in an unknown molecule [34,35]. Desorption ionization methods for large molecules usually produce several orders of magnitude more neutral molecules than ions; the latter could possibly be ionized by electrons with a concomitant increase in sensitivity. Recent photodissociation experiments of McIver et al. [32,33] using a 193 nm excimer laser indicates that these high-energy (6.4 eV) dissociations can
90
be highly selective. The excited species so produced could dissociate nonergodically so fast that energy is not randomized, which would appear to be especially advantageous for large ions containing many degrees of freedom. The process can be conveniently repeated for MS/MS/MS experiments ]331. SUMMARY
Obviously, many previous workers in ion cyclotron resonance and Fourier-transform mass spectrometry have appreciated many of these MS/MS capabilities for structural and analytical applications as well as for basic studies of ion chemistry [5,8-14,16-19,22-25,28-331. On the other hand, most of the recent MS/MS studies which have attracted such broad attention have been done on other than FTMS instruments. These have only been available commercially in the last few years and their first applications have been mainly in normal mass spectrometry. However, the advantages of FTMS for MS/MS are even more compelling and this technique appears to offer the best chance of making MS/MS nearly as routine a tool in the near future as mass spectrometry itself is today. ACKNOWLEDGMENTS
We thank R.B. Cody, Jr., M. Comisarow, J.J.P. Furlong, S. Ghaderi, D.P. Littlejohn, J.A. Loo, D.H. Russell, B.H. Wang, and E.R. Williams for valuable discussions, and the National Institutes of Health (grant GM16609) and the Ar,my Research Office (DAAG29-82-K-0179) for generous financial support. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983. R.W. Kondrat and R.G. Cooks, Anal. Chem., 50 (1978) 81A. F.W. McLafferty and F.M. Bockhoff, Anal. Chem., 50 (1978) 69. K. Levsen and H. Schwarz, Angew. Chem. Int. Ed. Engl., 15 (1976) 509. J.J. Zwinselman, N.M.M. Nibbering, B. Ciommer and H. Schwarz, in F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983, pp. 67-104. M.T. Cheng, G.H. Kruppa, F.W. McLafferty and D.A. Cooper, Anal. Chem., 54 (1982) 2204. M.T. Cheng, M.P. Barbalas, R.F. Pegues and F.W. McLafferty, J. Am. Chem. Sot., 105 (1983) 1510. M. Comisarow and A.G. Marshall, Chem. Phys. Lett., 25 (1974) 282. M.L. Gross and D.L. Rempel, Science, 226 (1984) 261. A.G. Marshall, Act. Chem. Res., 18 (1985) 316. D.A. Laude, Jr., C.L. Johlman, R.S. Brown, D.A. Weil and C.L. Wilkins, Mass Spectrom. Rev., 5 (1986) 107.
91 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
27 28 29 30 31 32 33 34 35
D.H. Russell, Mass Spectrom. Rev., 5 (1986) 167. F. Kaplan, J. Am. Chem. Sot., 90 (1968) 4483. T.M. Sack, D.A. McCrery and M.L. Gross, Anal. Chem., 57 (1985) 1290. R.A. Yost and C.G. Enke, Anal. Chem., 51 (1979) 1251A. R.B. Cody, R.C. Bumier, C.J. Cassady and B.S. Freiser, Anal. Chem., 54 (1982) 2225. D.L. Bricker, T.A. Adams, Jr. and D.H..Russell, Anal. Chem., 55 (1983) 2417. D.H. Russell, First European Symposium on FTMS, Metz, France, September 1985. R.B. Cody, Jr., I.J. Amster and F.W. McLafferty, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 6367. G.M. Neumann and P.J. Derrick, Org. Mass Spectrom., 19 (1984) 165. P.J. Todd, D.C. McGilvery, M.A. Baldwin and F.W. McLafferty, in F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983, pp. 271-286. J.J. Zwinselman, M. AIlemann and H.-P. Kellerhals, Information ICR, Spectrospin, Fallanden, Switzerland, 1984, Vol. III, August. A.G. Marshall, T.-C.L. Wang and T.L. Ricca, J. Am. Chem. Sot., 107 (1985) 7893. F.W. McLafferty, First European FTMS Symposium, Metz, France, September 1985. I.J. Amster, F.W. McLafferty, M.E. Castro, D.H. Russell, R.B. Cody, Jr. and S. Ghaderi, Anal. Chem., 58 (1986) 483. G.P. Jonsson, A.B. Hedin, P.L. Hakansson, B.U.R. Sundqvist, B.G.S. Save, P.F. Nielsen, P. Roepstorff, K.-E. Johansson, I. Kamensky and M.S.L. Lindberg, Anal. Chem., 58 (1986) 1084. R.D. Macfarlane, Anal. Chem., 55 (1983) 1247A. J.C. Tabet, R. Houriet and T. Gaumann, First European FTMS Symposium, Metz, France, September 1985. J.A. Loo, E.R. Williams, I.J. Amster, J.J.P. Furlong, B.H. Wang, F.W. McLafferty, B.T. Chait and F.H. Field, in preparation. R.B. Cody, Jr. and B.S. Freiser, Anal. Chem., 51 (1979) 547, R.C. Dunbar, in M.T. Bowers (Ed.), Gas Phase Ion Chemistry, Vol. 2, Academic Press, New York, 1979, pp. 181-220; Vol. 3,1984, pp. 129-166. R.T. McIver, Jr., W.D. Bowers, S.-S. Delbert and R.L. Hunter, J. Am. Chem. Sot., 106 (1984) 7288. W.D. Bowers, S.-S. Delbert and R.T. McIver, Jr., Anal. Chem., 58 (1986) 969. D.F. Hunt, C.N. McEwen and R.A. Upham, Anal. Chem., 44 (1972) 1291. S. Ingemann and N.M.M. Nibbering, J. Org. Chem., 48 (1983) 183.
International Journal of Mass Spectrometry and Zon Processes, 72 (1986) 85-91 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
TANDEM
FRED
FOURIER-TRANSFORM
W. McLAFFERTY
Chemistry Department, (First received
MASS SPECTROMETRY
** and I. JONATHAN
*
AMSTER
Cornell University, Ithaca, NY 148.53-1301 (U.S.A)
7 November
1985; in final form 30 June 1986)
ABSTRACT Advantages of the Fourier-transform mass spectrometer for tandem mass spectrometry (MS/MS) include the ability to perform consecutive mass separations (even MS”) in its one analyzer cell, unusually high resolution (lo6 at m/z 300) which is independent of ion kinetic energy, simultaneous (multichannel) detection of nearly all masses, sub-picomole sensitivity, high mass range ( >16000 daltons) without detector limitations, collisionally activated dissociation of ions of energies of volts to kilovolts with efficient daughter ion trapping, and flexible capabilites for ion/molecule reactions and ion photodissociation.
INTRODUCTION
The impressive recent advances in both Fourier-transform (FT) mass spectrometry and tandem mass spectrometry (MS/MS) make appropriate a review of their combined capabilities. MS/MS [l] can improve the specificity and sensitivity of MS with little sacrifice of its speed or accuracy. The first mass spectrometer (MS-I) can be used to separate molecular ions of mixture components produced by “soft” ionization, analogous to the chromatographic separation of GC/MS and LC/MS techniques. The separated molecular ion species can then be fragmented, for example by collisionally activated dissociation (CAD) or photodissociation. The resulting product ions separated in MS-II provide a secondary mass spectrum useful for structural characterization of the original molecular component [2,3] in much the same way that a normal mass spectrum is structurally characteristic of a pure sample. A second general use of MS/MS is to obtain additional structural information on pure compounds [4-71. “Hard” ionization, such as
Presented at the First European Symposium on Fourier-Transform Metz, France, 19 September 1985. ** To whom correspondence should be addressed. l
0168-1176/86/$03.50
0 1986 Elsevier Science Publishers
B.V.
Mass
Spectrometry,
86
that effected with 70 eV electrons, of a molecule produces fragment ions. Mass separation of these by MS-I and dissociation give MS-II mass spectra characteristic of the structures (even the stereochemistry [7]) of the individual molecular fragments, for which normal mass spectrometry with exact mass data only provides elemental composition assignments. However, most MS/MS instruments used to date have required a second mass analyzer, a substantial disadvantage in cost. In contrast, the ion cell of the FT mass spectrometer [8-121 can be used to select primary ions of a specific mass, dissociate or react these, and measure the secondary ions thus formed. Such an MS/MS capability with CAD was first demonstrated in 1968 [13] using the “double resonance” technique of ion cyclotron resonance spectrometry, the forerunner of FTMS. Although the unique capabilities of FTMS for normal mass spectrometry have been reviewed recently [9-121, both FTMS and MS/MS are progressing so rapidly that it appeared appropriate to review FTMS/FTMS and outline promising areas for future development . ION TRAPPING AND MEASUREMENT
FTMS employs “static” trapping of ions. Those moving at less than escape velocities orthogonal to the magnetic field (X and Y axes) are trapped in cyclotron orbits, while motions parallel to the field are contained by trapping plates with a small (e.g. 1 V) potential of the same charge. For mass measurement, all ions are accelerated with a broad-band rf frequency to orbits within the cell using the X-axis cell boundary plates. The Y-axis plates are then used to detect the orbiting packets of ions, each of which produces a complementary rf signal of magnitude proportional to the number of ions. Ions of many m/z values produce a corresponding multiplicity of overlapping frequencies; from these, the Fourier transform produces the mass spectrum, thus measuring all ions simultaneously. In contrast, scanning MS instruments must generate ions continuously, but at any instant can measure only those ions in a small mass range; to increase resolution, the width of this range must be decreased. Even for single-ion monitoring, the sensitivity of FTMS appears to be competitive with other MS instrumentation. For example, Gross and co-workers report [14] the detection of lo-i1 g of naphthalene at a resolution (m/Am) of 20 000 using multiphoton ionization with FTMS. MS/MS
For collisional dissociation (or ejection), ions of any chosen m/z value can be accelerated, providing an MS-I selection process. The secondary mass
87
spectrum can be measured by the same technique as that described for the primary ions, with the further advantage that the cyclotron frequency of an ion is only dependent on its mass-to-charge ratio (m/z) and the magnetic field, not on its kinetic energy. In contrast, magnetic and electrostatic analyzers often used as MS-II do not exhibit this kinetic energy independence and so the MS-II resolution is usually relatively poor ( < 100) because of the kinetic energy released in ion dissociation. Further, in such magnetic instruments with multi-kilovolt in acceleration, focusing of the secondary ions produced by CAD for introduction into MS-II is inefficient, with a collection angle of < l”, while FTMS ion trapping, especially in the X and Y axes, is quite efficient. For the low energy (lo-150 V) collisions in the triple quadrupole MS/MS instrument, the central rf-only quadrupole is similarly advantageous for focusing product ions scattered through larger angles [15]. CAD in magnetic instruments without special modification involves multi-kilovolt ion energies; with FTMS, such collision energies can be from the lowest values [16] up to the kilovolt range (2 keV for C,Hz’ ions in a 2.22 cm radius ion cell, 3T magnet [17]). Recent work by Russell [18] and Cody et al. [19] indicates that CAD of high mass (1000-2000 daltons) ions of sub-kilovolt energies may be much more effective than indicated by previous experiments with multi-kilovolt energies [20]. HIGH-RESOLUTION MS/MS
Tandem double-focusing mass spectrometers now available commercially can achieve resolving powers up to 100 000 in MS-I and 10 000 in MS-II (our homemade instrument, the first of this kind used in molecular studies [21], exhibits values of 50000 and 2000, respectively). FTMS, costing less than half the amount of these instruments, has achieved far higher resolution, such as 4 x lo6 at m/z 131 for MS-I [ll] and 330000 at m/z 145 in MS-II [22]. Such high FTMS resolution demands ion cell pressures near 10e9 torr, but these appear to be achievable conveniently by separate formation of ions outside the measurement cell; the dual cell developed by Nicolet Instruments appears to be particularly promising for this [ll]. Obviously, this resolving power makes possible a correspondingly impressive improvement in selectivity for MS/MS experiments. For example, consider the problem of the identification of the nerve gas sarin with high confidence. Of the 400 compounds of molecular weight 140 in our file of reference mass spectra, sarin is the only one which has the composition CqHi,,FOzP. Its mass differs by one part in 235000 from that of C,HsN,S, represented in the file by three compounds, and by l/141 000 from that of C,H,Cl, represented by 13 compounds (such as chloroxylenes). Thus, measurement of the mass of an
88
unknown m/z 140 ion with sufficient accuracy and resolution, in either MS-I or MS-II, depending on the experiment, would greatly increase the confidence in the identification of sarin, or prevent false alarms if the ions were actually of another composition. Selective rf excitation of primary ions at present can only be achieved with a resolving power of - 1000 [19], but the tailored excitation function developed by Marshall et al. [23] promises to give resolution in the selection process comparable with that achievable in the measurement process. FTMS resolution decreases linearly with increasing mass, so that 4 X lo6 resolution at m/z 100 predicts only unit resolution at m/z 20000. However, doublefocusing magnetic and time-of-flight instruments achieve only unit resolution or less above m/z - 5000.
MULTICHANNEL FTMS/FTMS
We recently suggested [24] another possible application of Marshall’s tailored excitation function to achieve the multichannel advantage simultaneously for MS-I and MS-II. At present, for the structural characterization of the multiplicity of fragment peaks in the spectrum of a large molecule, it is necessary to select and dissociate each primary ion individually, even though the measurement of the resulting MS-II spectrum can be done simultaneously with the multichannel advantage of FTMS. Thus, more sample must be ionized for each MS-II spectrum desired. If all primary ions are dissociated simultaneously, the resulting secondary spectrum shows the products of all primary ion dissociations together. To differentiate these, the CAD of all ions could be repeated, but using the tailored excitation function to dissociate differing proportions of the individual mass values. This could be done by reducing individual abundances through selective excitation to maximum orbits in which a fraction of the ions strike the cell walls. A Hadamard-like decoding function could then use the change in product ion abundances to assign precursor-daughter ion relationships. For example, if precursor ions of a particular m/z value are excited so that 50% are lost and all remaining ions are dissociated under the conditions of the original CAD spectrum, any daughter ions arising only from the selected m/z value should be reduced to 50% of their original abundance. The tendency of the most abundant CAD product ions to be formed by the loss of relatively small neutrals should reduce the degree of overlap in the simultaneous collisional dissociation of all fragment ions from a large molecule. Ambiguities remaining after one such modified CAD experiment could be clarified in a further experiment with appropriate abundance adjustments.
89 MASS RANGE
With rf circuitry giving sufficiently low frequencies, the mass range of FTMS should be virtually unlimited (> 100000) 191, although increasing the magnetic field increases both the resolution and sensitivity achievable at a ions at m/z 16241 have been detected by specific mass. Recently, Cs,& FTMS with good sensitivity [25]. The instrument used has a 3T magnet and this mass represents the limit of the rf excitation circuitry of the instrument. All masses from 100 to 16000 produced in a single ionization pulse were measured simultaneously. Dramatic progress has been made in the last few years (as described earlier [ 111) in several methods to ionize large molecules such as trypsin (molecular weight 23463) [26] by 252Cf plasma desorption [27]. In preliminary experiments using this method with FTMS, molecular ion species have been formed from leu-enkephalin [28] and gramicidin S [29]. As a recent example of FTMS/FTMS [19], laser ionization of - 10e8 g of the gramicidin D, a mixture of linear pentadecapeptides gave (M + K)+ ions of four components. Collisional dissociation of the m/z 1920-1921 ions gives a unit resolution CAD mass spectrum representing the major component. Differences corresponding to amino acid masses identified 11 residues from the N-terminal peaks and 8 from the C-terminal peaks, which with overlapping provided sequence information on 12 of the 15 amino acids of this component. Similar laser ionization of - lo-’ g of impure gramicidin S followed by CAD of its major (M + K)+ ion at m/z 1179 gave complete information on the amino acid sequence of this cyclic decapeptide. The resolution and mass accuracy make possible the clear distinction of the unusual amino acid ornithine, mass 114, from leucine, mass 113. OTHER METHODS
FOR CHARACTERIZING
PRIMARY
IONS
Besides both high- and low-energy CAD, other promising methods providing complementary structural information are made possible by the orders-of-magnitude longer ion residence times in FTMS. As pioneered using the forerunner technique of ion cyclotron resonance, these include ion/molecule reactions [13], electron bombardment [30], and laser photodissociation [31-331. Only our knowledge of ion/molecule chemistry limits the possibilities of the first, such as determining the H-D exchangeable hydrogen atoms in an unknown molecule [34,35]. Desorption ionization methods for large molecules usually produce several orders of magnitude more neutral molecules than ions; the latter could possibly be ionized by electrons with a concomitant increase in sensitivity. Recent photodissociation experiments of McIver et al. [32,33] using a 193 nm excimer laser indicates that these high-energy (6.4 eV) dissociations can
90
be highly selective. The excited species so produced could dissociate nonergodically so fast that energy is not randomized, which would appear to be especially advantageous for large ions containing many degrees of freedom. The process can be conveniently repeated for MS/MS/MS experiments ]331. SUMMARY
Obviously, many previous workers in ion cyclotron resonance and Fourier-transform mass spectrometry have appreciated many of these MS/MS capabilities for structural and analytical applications as well as for basic studies of ion chemistry [5,8-14,16-19,22-25,28-331. On the other hand, most of the recent MS/MS studies which have attracted such broad attention have been done on other than FTMS instruments. These have only been available commercially in the last few years and their first applications have been mainly in normal mass spectrometry. However, the advantages of FTMS for MS/MS are even more compelling and this technique appears to offer the best chance of making MS/MS nearly as routine a tool in the near future as mass spectrometry itself is today. ACKNOWLEDGMENTS
We thank R.B. Cody, Jr., M. Comisarow, J.J.P. Furlong, S. Ghaderi, D.P. Littlejohn, J.A. Loo, D.H. Russell, B.H. Wang, and E.R. Williams for valuable discussions, and the National Institutes of Health (grant GM16609) and the Ar,my Research Office (DAAG29-82-K-0179) for generous financial support. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983. R.W. Kondrat and R.G. Cooks, Anal. Chem., 50 (1978) 81A. F.W. McLafferty and F.M. Bockhoff, Anal. Chem., 50 (1978) 69. K. Levsen and H. Schwarz, Angew. Chem. Int. Ed. Engl., 15 (1976) 509. J.J. Zwinselman, N.M.M. Nibbering, B. Ciommer and H. Schwarz, in F.W. McLafferty (Ed.), Tandem Mass Spectrometry, Wiley, New York, 1983, pp. 67-104. M.T. Cheng, G.H. Kruppa, F.W. McLafferty and D.A. Cooper, Anal. Chem., 54 (1982) 2204. M.T. Cheng, M.P. Barbalas, R.F. Pegues and F.W. McLafferty, J. Am. Chem. Sot., 105 (1983) 1510. M. Comisarow and A.G. Marshall, Chem. Phys. Lett., 25 (1974) 282. M.L. Gross and D.L. Rempel, Science, 226 (1984) 261. A.G. Marshall, Act. Chem. Res., 18 (1985) 316. D.A. Laude, Jr., C.L. Johlman, R.S. Brown, D.A. Weil and C.L. Wilkins, Mass Spectrom. Rev., 5 (1986) 107.
91 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
27 28 29 30 31 32 33 34 35
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