BIOLOGICAL MASS SPECTROMETRY, VOL. 21, 413-419 (1992)

PERSPECTIVES

Hybrid Tandem Mass Spectrometers in Biological Research Simon J. Gaskell Center for Experimental Therapeutics, Baylor College of Medicine, Houston, Texas 77030, USA

INTRODUCTION The impact of tandem instruments on the field of mass spectrometry needs no emphasis. Tandem mass spectrometry (MS/MS) provides two essential experimental features: (i) the establishment of precursor/product ion relationships; (ii) the opportunity for enhancement of ion decompositions, usually following a collisional activation step. It is salutary to recall the long history of MS/MS for specific studies in ion chemistry,’ but the widespread application of the technique dates from the recognition of the potential of MS/MS for complex mixture analysis. A variety of instrument configurations have been htroduced and evaluated for MS/MS though rather few are widespread. The triple-quadrupole design was extremely influential in popularizing the use of MS/MS for analytical purposes, dating from the work of Yost and Enke in the late 1970s.’ At about the same time, Cooks3 and others emphasized the utility of the established reverse-geometry double-focusing sector instrument design4 for the analysis of complex mixtures using gas-phase decompositions. Resolution limitations prompted the construction of four-sector mass spect r o m e t e r ~ ;these ~ instruments have had a major impact (out of proportion to the number of installations), particularly in the area of biopolymer analysis. Double-focusing mass spectrometers comprising magnetic and electric sectors may be considered to be of ‘hybrid’ design, since they incorporate analyzers based on different separation principles. The term ‘hybrid‘, however, is most commonly used for combinations of sectors and quadrupole devices. The evaluation of hybrids for analytical purposes dates from the work of More than 150 instruments Cooks and have now been built by commercial manufacturers and installed worldwide, but the impact of these instruments in analytical research has not matched their availability. Are hybrid instruments over-rated by customers or under-used by their owners? It is not the intention of this ‘Perspective’ to provide a comprehensive review either of the fundamentals or of the applications of hybrid instruments; such treatments are available Rather the intention is to express an opinion concerning the role(s) of hybrid instruments in analytical research, with particular reference to the biological sciences. Two questions are addressed : (i) are the MS/ MS capabilities that are available on other instrument designs equalled or compro1052--9306/92/090413-07 $08.50

(01992 by John Wiley & Sons, Ltd.

mised in the hybrid configuration?; (ii) are there unique capabilities of hybrid instruments which are of analytical value? To answer these questions, applications of hybrid instruments will be discussed under the general headings of MS/MS, sequential mass spectrometry (MS3), and analyses which exploit the heterogeneous combination of ion analyzers. For convenience, most of the examples cited are from this laboratory; this is certainly not intended to imply exaggerated claims to pioneering contributions, and the work of other laboratories is duly acknowledged.

HYBRID INSTRUMENTS FOR MS/MS The majority of MS/MS analyses performed using hybrid instruments involve selection or scanning of precursor ion species using the double-focusing sector portion of the instrument, with decompositions occurring in a radio-frequency-only quadrupole (q), or other multipole device. Product ions are selected or scanned using a quadrupole mass filter (Q). The sector component of the instrument may be of ‘forward‘ (EB, where E = electric sector, and B = magnetic sector) or ‘reverse’ (BE) geometry. Thus, hybrid instruments may be designated as of EBqQ or BEqQ design. The implications of these different designs are discussed below. (It should be noted that the salient feature of the BEqQ design is that an electric sector follows the magnetic sector ; thus, apparently more complex configurations, such as EBEqQ, may be considered as equivalent to BEqQ for the purposes of this paper.) When the decompositions of selected precursor ions take place in 4, the energetics of collisionally activated decomposition (CAD) (if gas is introduced into q ) and the kinetics of the decompositions are similar to those which apply to MS/MS using a triple-quadrupole (QqQ). No formal comparisons have been made, however, of the performance of hybrid instruments and triple quadrupoles for the analysis of low-energy decompositions. It is not clear, for example, whether there are analytical implications to the differences between the instrument types with respect to the acceleration of the precursor ions and the time-scale of their selection. The MS/MS scan types which may be performed on triple quadrupoles (precursor, product and constant neutral loss) may similarly be carried out on hybrids. Received 22 April I992 Accepted 5 M a y 1992

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mlz

Figure 1. Product ion spectra obtained on a BEqQ instrument by collisional activation of isovalerylcarnitine [M + H]+ ions. (a) Low-energy (20 eV in the laboratory frame-of-reference) collisional activation (using argon) in q ; spectrum obtained by scanning Q. (b) High-energy (8 keV) collisional activation (using helium) in the 2nd FFR; spectrum obtained by scanning E . (c) Collisional activation as in (b); spectrum obtained by linked scanning of E and 0. Product ions of m/z 230 and 203 represent losses of CH, and C,H,', respectively, from the isovaleryl moiety and permit differentiation from the valeryl and 2-methylbutyryl isomers. (From Rapid Commun. Mass Spectrom. S. J. Gaskell and M. H. Reilly, 2,139. Copyright @ 1988 John Wiley & Sons, Ltd. Reprinted by permission of John Wiley & Sons, Ltd.).

Hybrid instrument control is somewhat more demanding (requiring, for example, careful calibration of the non-linear scan function of the magnet for constant neutral loss scanning). In practice, this is rarely limiting for single MS/MS experiments but the performance of multiple MS/MS scan types during a single analysis (such as gas chromatography/mass spectrometry (GC/MS) of a complex mixture) is more readily achieved using a triple quadrupole. The incorporation of a double-focusing mass spectrometer as MS1 permits (in addition to conventional high-resolution mass spectrometry for accurate mass determinations) the selection of precursor ions at enhanced resolution in MS/MS analyses. It was demonstrated several years ago, for example, that improved selectivity of detection during GC/MS/selected reaction monitoring analyses was achieved by selection of the precursor ion with modestly increased resolution (m/m 5000).' This facility has, however, been exploited very little despite the acceptance of selected reaction monitoring as a quantitative trace analysis technique. The MS/MS capabilities associated with the doublefocusing mass spectrometer are not compromised in the hybrid instrument. Thus, instruments of either EBqQ or BEqQ geometry may be used for the study of decompo-

sitions in the first field-free region (1st FFR) between the ion source and the first sector. Product ion resolution is generally satisfactory but effective precursor ion resolution is poor. BEqQ instruments may also be used for mass-analyzed ion kinetic energy spectroscopy (MIKES), involving the study of decompositions in the 2nd FFR (between B and E). In this instance, the resolution problem is reversed; precursor ion resolution is satisfactory but the mass resolution of product ions is poor. If product ions formed in the 2nd FFR are transmitted to the quadrupole assembly, however, and E and Q are scanned in unison," the mass resolution of product ions is determined by the quadrupole mass filter and may therefore be impr~ved.".'~ The principal advantage of analyses of decompositions taking place in the sector portion of the hybrid instrument is that high-energy collisional activation may thereby be incorporated. Figure 1 illustrates the benefit of high-energy CAD with E / Q linked scanning in the analysis of an a~ylcarnitine.'~ Product ions characteristic of the acyl moiety result only from high-energy decompositions; precise mass assignments are facilitated by improved product ion resolution. In practice, the achievement of a satisfactory E / Q linked scan (and associated linkages of float potentials)

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m/z Figure 2. Mass spectrometric analyses (using a hybrid instrument) of an adipokinetic hormone derived from Drosophila rnelanogaster. (a) Conventional FAB mass spectrometric analysis, using an estimated 50 pmol of sample. (b) Product ion spectrum derived from lowenergy CAD of putative [M + H]+ ions, rn/z 975; an estimated 150 pmol of sample was used. (c) Product ion spectrum derived from low-energy CAD of [M + H]+ ions, rn/z 975, of the synthetic peptide, pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH,. (Reproduced with permission from ref. 20.)

across a broad kinetic energy/mass range may be diEcult. Furthermore, variations in the extent of kinetic energy loss accompanying a collision which results in fragmentati~n'~ will affect the optimum link between the E and Q scans. Boyd and coworkers adopted two innovative approaches to address these issues. In the first," E and Q are scanned concurrently but asynchronously; an accumulation of spectra recorded in this way is equivalent to an optimized E/Q linked scan. In a second approach," a modulation is superimposed on the E scan to allow for variations in the optimum E/Q link and for experimental imprecision. In this laboratory, an alternative approach has been taken in those instances where the requirement for improved mass resolution of high-energy CAD product ions is limited to a few ions in each spectrum." Selected portions of the MIKE spectrum are examined in detail by Q scanning at fixed E, or E scanning at fixed Q. The net result is again improved definition of the m/z ratios of the product ions of interest. The improved resolution of the product ions of highenergy CAD by use of the quadrupole filter in a BEqQ instrument is achieved at a significant cost in transmission. The approach is not, therefore, equivalent to analyses using a four-sector instrument, in which the transmission of product ions is improved by the doublefocusing configuration of MS2. Moreover, incorporation of photodiode arraysIg for the detection of product ions using four-sector instruments provides further dividends with respect to sensitivity. Thus, the facilities for analysis of the product ions of high-energy CAD accomplished using a BEqQ instrument should be

recognized as a significant improvement over the MIKES technique and considered an important analytical tool in evaluating the need for high-energy CAD in a particular analytical problem. These facilities for the study of high-energy CAD should not, however, be considered as competitive with four-sector capabilities. Both low and high-energy CAD have been found useful in structural characterization of peptides using MS/MS. Figure 2 shows an example of the analysis of a peptide of biological origin using low-energy CAD on a hybrid instrument." Conventional mass spectrometry [Fig. 2(a)] was informative only with respect to the definition of molecular mass. Extensive sequence information was observed in the product ion spectrum [Fig. 2(b)] derived by CAD of putative [M HI+ ions, and the characterization was strengthened by a comparison with equivalent data for the synthetic peptide [Fig. 2(c)]. The low-energy CAD data alone, however, were not able to define the total sequence. (The product ion spectrum does not, for example, permit a distinction between leucine and isoleucine residues. In this example, an independent amino acid analysis indicated the presence of leucine.) Several comparative studies of lowenergy CAD (using hybrid instruments) and high-energy CAD (using four-sector instruments) have demonstrated a greater information content of product ion spectra of higher-mass protonated peptides ( M , > 1OOO) obtained following high-energy CAD21-23 (including the distinction of leucine and isoleucine residue^).^' This generalization is likely to remain valid despite recent evidence from this laboratory2' that an improved understanding of the parameters determining peptide ion fragmenta-

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tions may be observed in the 1st FFR and in q ; thus, for tion substantially increases the structural information example, appropriate setting of B and E transmits a that may be obtained from low-energy decomposition chosen product of a specific parent to the quadrupole spectra. decomposition region (q), and Q may be scanned to Hybrid instruments possess a clear advantage over provide a spectrum of second-generation product ions. four-sector instruments with respect to other MS/MS A second approach is possible with instruments of experiments-precursor ion and constant neutral loss BEqQ design: selection of the precursor ion using B scanning. These modes of analyses are of critical impormay be followed by selection of the first-generation tance in such applications as screening for xenobiotic product ion (formed in the 2nd FFR) using E. Q is metabolites, as a number of studies have demonstrated, using both and t r i p l e - q ~ a d r u p o l e ~ ~ ~again ~ ~ scanned to provide a spectrum of secondgeneration product ions formed in q. (Third-generation instruments. In this laboratory, for example,z6 the presproduct ion scanning is also possible on the BEqQ ence of a glutathione conjugate metabolite of 2instrument if decompositions are examined in 1st FFR, furamide in rat bile was established using both constant 2nd FFR and q.)32 Figure 3 shows an example of neutral loss and precursor ion scanning on a hybrid second-generation product ion scanning in the study of instrument; additional structural detail was obtained by the formation of a C-terminal rearrangement product subsequent scanning of product ions derived from the from peptide [M + HI' ions.33 The spectrum of previously recognized precursor. Establishment of consecond-generation product ions, formed via the stant neutral loss scanning on a four-sector instrument rearrangement ion, was compared with the spectrum of would be extraordinarily complex and has not been first-generation product ions obtained by MS/MS demonstrated. Precursor ion scanning has been analysis of the protonated peptide lacking the Creported3' but not widely applied. The breadth of the terminal amino acid residue. The close similarity of the scan is dependent on the energy acceptance of E,; thus, spectra provided strong evidence that the structure of the recent demonstration3' of extended range precursor the rearrangement ion was that of the protonated trunion scanning using a four-sector instrument incorpocated peptide. rating an inhomogeneous E, with very broad energy The two approaches to MS3 have different implicaacceptance may suggest an increased application of tions with respect to the effective resolutions of precurfour-sector instruments in this area. sor and first-generation product ions. When 1st FFR To summarize the MS/MS capabilities of hybrid decompositions are examined, the effective resolution of mass spectrometers in comparison with alternative precursor ions is low (as noted above in discussion of tandem instrument types, the following points can be 1st FFR decompositions in MS/MS analyses). When made. (i) For the study of low-energy decompositions, 2nd FFR decompositions are studied, limited mass the capabilities are formally equivalent to those of triple resolution is observed for the first-generation products quadrupoles, though rigorous comparisons are lacking (as in MIKES). In this laboratory's experience, unit of the quality of data obtained using the two instrument resolution of both precursor and first-generation types. Some ease of instrument control is sacrificed with product ions (in addition to second-generation product the hybrid. (ii) For the study of high-energy CAD, a ions) is only rarely required in the same analysis, so that significant enhancement of capabilities is achieved by one or other of the approaches to MS3 is applicable. the addition of the quadrupole assembly to the doubleThe MS3 capabilities of hybrid instruments have been focusing instrument; these capabilities do not, however, described in terms of sequential product ion scanning match those of four-sector instruments. (iii) The key since this mode is arguably the simplest conceptually. advantage of the hybrid instrument is versatility, with As the work of Cooks, Enke and coworkers'2~34has respect to both the conditions of precursor ion activaemphasized, however, the number of possible scantion and decomposition, and the accessible scan types. modes increases markedly with stages of MS". Recent experience suggests that the reaction intermediate scan (an MS3 scan) is of particular value. The modes of operation possible with the BEqQ and EBqQ hybrids are HYBRID INSTRUMENTS FOR SEQUENTIAL analogous to those described for sequential product ion MASS SPECTROMETRY (MS3) scanning. Such scans have been used in this laboratory to elucidate the origins of 'mid-chain' fragments derived from protonated peptides (Fig. 4).35 The discussion above has emphasized the versatility MS3 analyses are not possible with triple-quadrupole associated with the presence of more than one possible instruments but may be achieved with pentaquadrupodecomposition region in hybrid instruments. The les (though the latter are not available as commercial second implication of this design is that multiple stages instruments). Cooks and coworkers have demonstrated, of decomposition may be observed, permitting sequenfor example, reaction intermediate scanning for the clartial mass spectrometric experiments. In general, the ification of sequence information for small peptides specificity of analysis increases with additional stages of obtained using a p e n t a q ~ a d r u p o l e .By ~ ~ selecting the MS/MS. Thus, for example, the structure of a firstgeneration product ion can be probed by subjecting it [M HI' ion as the precursor and the protonated Cto further collisional activation and analyzing products terminal amino acid as the final product, the reaction in a second-generation product ion scan. intermediate scan yielded a spectrum including only CThere are significant differences between EBqQ and terminal fragment ions, representing a considerable simBEqQ instruments with regard to the performance of plification of the MS/MS product ion scan. MS3 a n a l y ~ e sFor . ~ both designs, sequential decomposiIn principle, MS"+ analyses may be performed using

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mlz 887

1060

904

mlz Figure 3. Product ion spectra of metastable decompositions of ions of m/z 904 corresponding to (a) des-Arg9-bradykinin [M + H I + ions formed in the FAB ion source, and (b) [BE' + OH]+ ions formed in the 2nd FFR by decomposition of bradykinin [M + H I + ions. Assignments are made according to the nomenclature of Roepstorff and F ~ h l m a n . ~ The * close similarity of the two product ion spectra provides strong evidence for the identity of the C-terminal rearrangement ion (m/z 904) derived from bradykinin [M + H]+ and protonated des-Arggbradykinin. (Reprinted by permission of the publisher from G. C. Thorne, K. 0.Ballard and S. J. Gaskell, J. Am. SOC.Mass Spectiom. 1, 249. Copyright 1990 by Elsevier Science Publishing Co., Inc.). [M+H]+

Yn"

1191

966

Substance P (2-11) (PKPQQFFGLM-NH2) J

IM + H-NW] + 1174

KINFIlC ENERGY (eV)

Figure 4. Reaction intermediate scan of des-Arg'-substance P (PKPQQFFGLM-NH,, using the single-letter code to represent the amino acid sequence), corresponding to the fragmentation pathway, [M + H]+ + intermediates + (B9Y8)7'. The precursor ion was selected using 19 and was allowed to fragment in the 2nd FFR. First-generation product ions were allowed to fragment further in q. 0 was set to transmit second-generation product ions of m/z 818, corresponding to (BgYs)7'. Assignments are made according to the nomenclature of Roepstorff and F ~ h l m a n(Reproduced .~~ with permission from Ref. 35).

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a multisector instrument incorporating n field-free regions. While this has been d e m ~ n s t r a t e d ,analytical ~~ applications have been sparse. Experience in this laboratory with sequential mass spectrometric analyses using a BEqQ instrument has prompted collaborative studies to evaluate several new analytical modes using a foursector instrument incorporating an inhomogeneous second electric sector (with very broad energy acceptance) and an array detector.38 Performance was substantially improved by use of the array detector; nevertheless, comparison of sequential product ion scan data with equivalent results from a BEqQ instrument indicated that the signal/noise ratio achieved was a function of the energetics and kinetics of the decomposition processes as well as the sensitivity of detection of the final product ions. Reaction intermediate scanning was also established. MS" experiments such as those performed on hybrid instruments have illustrated the potential of this suite of techniques in biological applications. More widespread application of MS" analyses may ultimately be associated with alternative instrument types, such as the ion trap mass ~ p e c t r o m e t e r .Nevertheless, ~~ the versatility of hybrid mass spectrometers with respect to the choice of decomposition conditions suggests a lasting value for development work on these instruments.

EXPLOITING THE HYBRID'S HETEROGENEOUS COMBINATION OF ION ANALYZERS In this section, we consider explicitly the advantages arising from the incorporation in the hybrid design of ion analysers based on different separation principles (presaged by the discussion of E / Q linked scanning above). The BEqQ instrument incorporates sequential separations of ions according to momentum/charge (in B), kinetic energy/charge (in E), and mass/charge (in Q). A particularly clear example (albeit from outside the area of biological research) of the benefit of this combination comes from the work of Ross and coworkers on the products of high-energy collision of the fullerene ion, C,+,, with neutral helium.40 Assessment of the postulated formation of a helium adduct (He@C&) had unusual analytical requirements since the product was expected to possess a decreased kinetic energy/charge ratio concomitantly with an increased mass/charge ratio. Accordingly, Czo (m/z 720) ions were selected using B in a BEqQ instrument and were subjected to collision with helium in the 2nd FFR. Q was set to transmit m/z 724 (the mass/charge ratio of the putative adduct) and the electric sector was scanned. A clear signal was observed corresponding to an ion kinetic energy about 50 eV lower than that of the precursor ion, consistent with efficient uptake of the center-ofmass collision energy.40 A recent investigation by Ballard41 in this laboratory has exploited the different analyzers of a hybrid instrument to probe the origin of the pronounced tailing signal generally observed on the low-energy side of the precursor ion peak in fast atom bombardment (FAB)/

MIKES analyses of metastable decompositions. The tail was eliminated when the analysis was performed in the mass-deconvoluted MIKES mode, where detection followed transmission through the quadrupole assembly with Q set to the mass/charge ratio of the precursor ion. Thus, the major contributors to the tailing signal were not, in this instance, precursor ions with kinetic energies reduced by collision in the 2nd FFR. When components of the tailing signal were transmitted by appropriate setting of E, and Q was scanned, ions of mass/charge ratio higher than that of the selected precursor were detected. Transmission of such ions through the magnetic sector required that they possessed the same momentum/charge ratio as the intended precursor ions so that their higher mass/charge ratio necessarily indicated a velocity lower than that predicted on the basis of acceleration associated with the full source potential. The production of ions emerging from the ion source with lower-than-expected velocities was further substantiated by a series of momentum/charge scans (using B) of energy-selected (using E ) and mass-selected (using Q) precursor ions. Such experiments are unique to the hybrid instrument of BEqQ geometry. Collectively, the data suggested the hypothesis that ions emerging from the FAB source included a proportion with lower-thanexpected kinetic energy. This was attributed to an origin by desolvation in the acceleration region of ions desorbed from the FAB target as solvated species.

CONCLUSIONS The major conclusion of this summary of the capabilities of hybrid mass spectrometers for MS/MS is that these instruments represent highly versatile platforms for a broad range of MS/MS and MS3 experiments. Thus, for the laboratory with a limited instrumentation budget, the hybrid instrument (particularly of BEqQ geometry, or equivalent) may represent a cost-effective choice, while recognizing that other, less versatile instruments may offer superior performance for particular applications. More generally, the facility for evaluation on a single instrument of a variety of MS/MS approaches to the solution of a particular analytical problem will permit an appropriate choice of alternative instrument for extended application. Finally, the heterogeneous combination of analyzers in the hybrid design provides unparalleled capabilities for the characterization of ions and accordingly ensures a unique role for these instruments in analytical research.

Acknowledgements Contributions from this laboratory to the evaluation and exploitation of hybrid instruments have resulted from the expertise and commitment of past and present colleagues. In particular, I am grateful to the following: 0. Burlet, Dr K. D. Ballard, R. S. Orkiszewski, M. H. Reilly and Dr G. C. Thorne. Financial support for this aspect of the laboratory's work has been received from the National Institutes of Health and Glaxo, Inc.

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