Anal. Chem. 2004, 76, 7346-7353
Multiplexed MS/MS in a Quadrupole Ion Trap Mass Spectrometer Jonathan Wilson and Richard W. Vachet*
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
A multiplexing method for performing MS/MS on multiple peptide ions simultaneously in a quadrupole ion trap mass spectrometer (QITMS) has been developed. This method takes advantage of the inherent mass bias associated with ion accumulation in the QITMS to encode the intensity of precursor ions in a way that allows the corresponding product ions to be identified. The intensity encoding scheme utilizes the Gaussian distributions that characterize the relationship between ion intensities and rf trapping voltages during ion accumulation. This straightforward approach uses only two arbitrary waveforms, one for isolation and one for dissociation, to gather product ion spectra from N precursor ions in as little as two product ion spectra. In the example used to illustrate this method, 66% of the product ions from five different precursor peptide ions were correctly correlated using the multiplexing approach. Of the remaining 34% of the product ions, only 6% were misidentified, while 28% of the product ions failed to be identified because either they had too low intensity or they had the same m/z ratio as one of the precursor ions or the same m/z ratio as a product ion from a different precursor ion. This method has the potential to increase sample throughput, reduce total analysis times, and increase signal-to-noise ratios as compared to conventional MS/MS methods. Several means of sensitively obtaining structural information for components in complex mixtures using mass spectrometry (MS) combined with separation techniques have been developed. Liquid chromatography (LC) or capillary electrophoresis is often coupled with tandem mass spectrometry (MS/MS) to gather the desired structural information.1-12 For protein analyses, another common experimental procedure is to perform 2D gel electro* To whom correspondence should be addressed. Tel: (413) 545-2733. Fax: (413) 545-4490. E-mail:
[email protected]. (1) Yates, J. R., III; McCormack, A. L.; Link, A. J.; Schieltz, D.; Eng, J.; Hays, L. Analyst 1996, 121, 65R-76R. (2) Figeys, D.; van Oostveen, I.; Ducret, A.; Aebersold, R. Anal. Chem. 1996, 68, 1822-1828. (3) McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R., III. Anal. Chem. 1997, 69, 767-776. (4) Jin, X.; Kim, J.; Parus, S.; Lubman, D. M.; Zand, R. Anal. Chem. 1999, 71, 3591-3597. (5) Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III. Anal. Chem. 2000, 72, 757-763. (6) Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Anal. Chem. 2000, 72, 4266-4274. (7) Yates, N.; Wislocki, D.; Roberts, A.; Berk, S.; Klatt, T.; Shen, D.-M.; Willoughby, C.; Rosauer, K.; Chapman, K.; Griffin, P. Anal. Chem. 2001, 73, 2941-2951.
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phoresis to separate the complex mixtures, and then after removal and digestion of protein spots, MALDI-MS13,14 or LC-ESI-MS/ MS13,14 is used to gather further information. Because 2D gel separations can be time-consuming, an alternative approach has been developed by Yates and co-workers.3,5,12 This approach relies upon enzymatic digestion of a mixture of proteins, followed by LC separation and peptide sequencing by MS/MS. Proteins are identified from protein database searches that use peptide sequence tags15 generated from MS/MS data. While this approach has been quite successful, coelution of peptides and components with low abundance can lead to time-consuming repeats of experiments. Also, for very complex mixtures, the ability to identify multiple proteins depends on how many peptides can be subjected to MS/MS during the course of the LC separation. Recently, these issues have been addressed by reducing chromatographic flow rates and utilizing “peak parking” schemes to increase the number of MS/MS analyses that can be performed on coeluting components.6,16-19 These peak parking schemes have successfully increased the number of peptide ions that can be subjected to MS/MS, but they also increase analysis time, decrease separation efficiency, and decrease MS sensitivity. The ability to increase the throughput of MS/MS experiments might obviate the need for such peak parking strategies and thus increase sensitivity and decrease analysis time. To increase MS/MS throughput, we have developed a multiplexed MS/MS approach on a quadrupole ion trap mass spectrometer (QITMS) whereby several ions of different mass-to(8) Li, L.; Masselon, C. D.; Anderson, G. A.; Pasˇa-Tolic´, L.; Lee, S.-W.; Shen, Y.; Zhao, R.; Lipton, M. S.; Conrads, T. P.; Tolic´, N.; Smith, R. D. Anal. Chem. 2001, 73, 3312-3322. (9) Berger, S. J.; Lee, S.-W.; Anderson, G. A.; Pasˇa-Tolic´, L.; Tolic´, N.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2002, 74, 4994-5000. (10) Thompson, A.; Scha¨fer, J.; Kuhn, K.; Kienle, S.; Schwarz, J.; Schmidt, G.; Johnstone, R.; Neumann, T.; Hamon, C. Anal. Chem. 2003, 75, 1895-1904. (11) Ivanov, A. R.; Zang, L.; Karger, B. L. Anal. Chem. 2003, 75, 5306-5316. (12) Venable, J. D.; Yates, J. R., III. Anal. Chem. 2004, 76, 2928-2937. (13) Lim, H.; Eng, J.; Yates, J. R., III; Tollaksen, S. L.; Giometti, C. S.; Holden, J. F., Adams, M. W. W.; Reich, C. I.; Olsen, G. J.; Hays, L. G. J. Am. Soc. Mass Spectrom. 2003, 14, 957-970. (14) Terry, D. E.; Umstot, E.; Desiderio, D. M. J. Am. Soc. Mass Spectrom. 2004, 15, 784-794. (15) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (16) Moore, R. E.; Licklider, L.; Schumann, D.; Lee, T. D. Anal. Chem. 1998, 70, 4879-4884. (17) Gallis, B.; Corthals, G. L.; Goodlett, D. R.; Ueba, H.; Kim, F.; Presnell, S. R.; Figeys, D.; Harrison, D. G.; Berk, B. C.; Aebersold, R.; Corson, M. A. J. Biol. Chem. 1999, 274, 30101-30108. (18) Zhou, J.; Rusnak, F.; Colonius, T.; Hathaway, G. M. Rapid Commun. Mass Spectrom. 2000, 14, 432-438. (19) Meiring, H. D.; van der Heeft, E.; ten Hove, G. J.; de Jong, A. P. J. M. J. Sep. Sci. 2002, 25, 557-568. 10.1021/ac048955d CCC: $27.50
© 2004 American Chemical Society Published on Web 11/18/2004
charge (m/z) ions are subjected to MS/MS simultaneously instead of analyzing one m/z at a time. The multiplexing method should increase sample throughput, and signal-to-noise ratios should be enhanced due to the multiplexing advantage. The obvious challenge that arises with any multiplexing MS/MS approach is in the matching of product ions to their corresponding precursor ions, which is no longer simply accomplished because more than one precursor ion is isolated and dissociated. Consequently, another means of encoding the product ions that originate from each precursor ion is needed. Several methods for overcoming this encoding problem and enabling multiplexed MS/MS methods have been described. Most of these methods have been implemented on Fourier transform ion cyclotron resonance (FTICR) mass spectrometers. One approach was developed by McLafferty and co-workers20 and relies on repeating MS/MS experiments on appropriate subsets of precursor ions in order to assign product ions to the corresponding precursor ions. With this method, N spectra are required to effectively obtain the product ion spectra for N precursor ions. A Hadamard transform (HT) is then used to deconvolute the resulting data and thus determine the precursor ions from which each product ion derives. A second multiplexed MS/MS approach relies on FT techniques similar to those used in 2D NMR experiments. In this method, precursor ions are excited into their cyclotron orbits by an rf excitation pulse, which is then followed by an rf de-excitation pulse after a specific delay time (td). This de-excitation pulse changes the abundance of specific precursor ions according to the phase difference, which is dependent on the precursor ion m/z ratio. The product ion spectra are then recorded as a function of td, and the FT of this function allows the product ions to be related to their corresponding precursor ions by their abundance changes. Two variations to this approach have been described, one by Bodenhausen and Gaumann21 and another by Marshall and co-workers.22 Both the HT and 2D approaches to multiplexing on a FTICR are too slow for on-line coupling and require large amounts of sample to implement offline. Both methods, though, do provide signal-to-noise ratio enhancements. A third approach developed by Smith and co-workers allows multiplexed MS/MS to be performed in a single spectral acquisition. This approach relies on database searching and the high mass accuracy and resolution capabilities of the FTICR.8,23 Product ion spectra of N precursor ions are collected in one spectrum. The precursor ion m/z ratios and the resulting product ion m/z ratios are then compared to a database. Hypothetical product ion spectra of peptide fragments from this protein database are generated and compared to the experimentally determined product ion spectrum. The high mass accuracy of the FTICR mass spectrometer allows the number of potential peptide precursor ions to be reduced, and the hypothetical product ion spectra of the “candidate” peptides can be compared to the experimental data. Ultimately, assignment of the product ions to their respective (20) Williams, E. R.; Loh, S. Y.; McLafferty, F. W.; Cody, R. B. Anal. Chem. 1990, 62, 698-703. (21) Pfa¨ndler, P.; Bodenhausen, G.; Rapin, J.; Walser, M.-E.; Ga¨umann, T. J. Am. Chem. Soc. 1988, 110, 5625-5628. (22) Ross, C. W., III; Guan, S.; Grosshans, P. B.; Ricca, T. L.; Marshall, A. G. J. Am. Chem. Soc. 1993, 115, 7854-7861. (23) Masselon, C.; Anderson, G. A.; Harkewicz, R.; Bruce, J. E.; Pasˇa-Tolic´, L.; Smith, R. D. Anal. Chem. 2000, 72, 1918-1924.
precursor ions is accomplished by matching the peptide mass tag and the hypothetical product ion spectra with the experimentally determined data. While this multiplexed method can be used with on-line separations, only peptides that exist in a database can be identified. An additional drawback to FTICR mass spectrometers when it comes to multiplexed MS/MS experiments is their relatively high cost. Consequently, a multiplexed MS/MS method based on a less expensive and more widely accessible mass spectrometer would be beneficial. The trapping nature, the relatively low cost, and the ease, speed, and high efficiency with which MS/MS analyses can be performed on a QITMS make this mass spectrometer a viable candidate for multiplexed MS/MS experiments. Indeed, preliminary work by Glish and co-workers24,25 has shown the potential of QITMS for such analyses. By accumulating different precursor ions in the ion trap for different time periods using arbitrary waveforms, these researchers have demonstrated that precursor ions can be encoded by their intensities and product ions can be correlated accordingly. A drawback with this approach is that several complex arbitrary waveforms are required during ion accumulation, and the required number of waveforms increases as the number of precursor ions increases. Additional arbitrary waveforms complicate the experiment and increase analysis time. In this work, we describe a faster and simpler method for performing multiplexed MS/MS on a QITMS, in which encoding of the precursor ions is accomplished by relying on the inherent mass bias exhibited by this device during ion accumulation. EXPERIMENTAL SECTION Instrumentation. All experiments were carried out in a modified Bruker Esquire∼LC quadrupole ion trap mass spectrometer (Bruker Daltonics Inc., Billerica, MA). Isolation and dissociation waveforms were output from a Wavetek model 39 arbitrary waveform generator (Fluke Corp., Everett, WA) and were applied to the entrance end cap of the Esquire. A model DG535 digital delay pulse generator (Stanford Research Systems, Inc., Sunnyvale, CA) was used to provide the appropriate delay time between the trigger pulse provided by the Esquire hardware and the timing required during the ion trap scan function for the isolation and dissociation waveforms. The waveforms provided by the Esquire hardware, which are normally applied to the exit end cap, were used during ion acquisition. Stored Waveform Inverse Fourier Transform (SWIFT) Waveforms. SWIFT waveforms were constructed in LabView 6.0 (National Instruments Corp., Austin, TX) and downloaded via a GPIB interface card to the waveform generator. Isolation waveforms contained 16 384 points and were output at 10 Vp-p. During ion isolation when the external arbitrary waveforms were applied, the Esquire isolation waveforms were turned off by setting the Coarse High, Fine High, and Fine Low ion isolation settings all to 0 in the software. Dissociation waveforms contained 8192 points and were output at 2 Vp-p. The dissociation waveforms were substituted in for the normal Esquire dissociation waveforms by setting the fragmentation amplitude to 0 Vp-p in the Esquire (24) Ray, K. L.; Glish, G. L. Proceedings of the 48th ASMS Conference on Mass Spectrometry and Allied Topics; Long Beach, CA, June 11-15, 2000. (25) Cunningham, C., Jr.; Ray, K. L.; Glish, G. L. Proceedings of the 52nd ASMS Conference on Mass Spectrometry and Allied Topics; Nashville, TN, May 2327, 2004.
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software. Isolation and dissociation waveforms were output at 1 MHz, and they were all constructed with 10 m/z unit widths (i.e., 5 m/z units on either side of the precursor ions of interest). Reagents. Cys-kemptide, leucokinin V, angiotensin III antipeptide, and [D-Ala2, D-Pro4, Tyr5]-β-casomorphin (1-5) amide were obtained from the American Peptide Co., Inc. (Sunnyvale, CA). All other peptides and proteins, including bursin, TRH-Gly, angiotensinogen fragment 11-14, leucine enkephalin, methionine enkephalin, bradykinin fragment 2-9, angiotensin II, bradykinin, Tyr-bradykinin, angiotensin I, bombesin, and cytochrome c, were obtained from Sigma Chemical Co. (St Louis, MO). All peptides, whether in solution as part of a mixture or by themselves, were dissolved in 1:1 methanol/water with 3% acetic acid to concentrations of 10 µM. MS Conditions. Ion optics were kept at the following settings for the experiments involving the peptide ions. Skimmers 1 and 2 were 30 and 10 V, respectively, and the capillary exit offset was set to 80 V. The octopole ion guides were set at 2.70 V, with the octopole ∆ at 2.50 V and the octopole rf at 175 Vp-p. Lenses 1 and 2 were -5 and -60 V, respectively. For the experiments with cytochrome c, the conditions used for the ion optics were as follows. Skimmers 1 and 2 were 10 and 6 V, and the capillary exit offset was 68 V. The octopole ion guides were set at 2.62 V, with the octopole ∆ at 2.40 V and the octopole rf at 175 Vp-p. Samples were introduced into the electrospray source at a flow rate of 1 µL/min. The helium bath gas pressure in the vacuum system was maintained at a pressure of 1.34 × 10-4 mbar for all experiments. Data Processing. All spectra were processed using the Bruker Data analysis software and exported as ASCII files. These files were then processed within Microcal Origin 6.0 (Northampton, MA) to obtain the Gaussian fits and ratio spectra. RESULTS AND DISCUSSION A commonly considered disadvantage of quadrupole ion trap mass spectrometers is the limited and variable m/z range over which ions can be efficiently accumulated from external ion sources. This fact is illustrated by the spectra in Figure 1, which shows how the intensities of multiply charged cytochrome c ions change as the rf voltage applied to the ring electrode during ion accumulation is varied. Changing the rf voltage such that the lowmass cutoff (LMCO) increases by 10 Da greatly influences the intensity of the ions in the mass spectrum. Whereas in Figure 1a charge states ranging from +20 to +15 are seen when the LMCO is 41 Da (rf accumulation voltage ∼257 Vp-p), Figure 1b shows charge states ranging from +18 to +13 when the LMCO is increased to 51 Da (rf accumulation voltage ∼320 Vp-p). If the normalized ion intensity of the +15 charge state is plotted over a range of LMCO values, it is clear that the intensity of this ion displays a Gaussian distribution as a function of the LMCO used during accumulation (Figure 2a). A similar distribution of ion intensities as a function of LMCO has been reported previously.26-28 Plotting the normalized intensities for select charge states of cytochrome c shows that other ions also display Gaussian (26) McLuckey, S. A.; Glish, G. L.; Asano, K. G. Anal. Chim. Acta 1989, 225, 25-35. (27) Louris, J. N.; Amy, J. W.; Ridley, T. Y.; Cooks, R. G. Int. J. Mass Spectrom. Ion Processes 1989, 88, 97-111. (28) Kofel, P. In Practical Aspects of Ion Trap Mass Spectrometry; March, R. E., Todd, J. F. J., Eds.; CRC Press: New York, 1995; Vol II, pp 51-87.
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Figure 1. Mass spectra of cytochrome c at LMCO of (a) 41 and (b) 51 Da.
Figure 2. (a) Normalized ion intensity for the +15 charge state of cytochrome c as a function of LMCO and (b) normalized ion intensities for different charge states of cytochrome c as a function of LMCO.
distributions and that the centers of these distributions increase with increasing m/z (Figure 2b). Further study was undertaken with a series of peptide ions to observe the relationship between ion intensity and LMCO during ion accumulation. Just as for the protein ions, the peptide ions display a Gaussian distribution in ion intensity as a function of
Figure 4. Illustration of how the intensity encoding scheme is applied. The lines labeled 1° and 2° indicate the LMCO values at which precursor ions are accumulated in the primary and secondary spectra, respectively.
In eq 1, w is related to the width of the distribution and c is the center of the distribution. The fractional change in ion intensity (F1°-2°) in going from the primary spectrum to the secondary spectrum is obtained by evaluating the Gaussian expression for two different x (or LMCO) values and taking their ratio (eq 2). Figure 3. (a) LMCO leading to maximum peptide ion intensity as a function of peptide ion m/z. (b) Widths of the Gaussian distributions as a function of m/z.
LMCO. Furthermore, a linear relationship exists between the optimum LMCO during ion accumulation (i.e., Gaussian center) and peptide ion m/z (Figure 3a). Such a linear relationship between the optimum LMCO and ion m/z is not surprising as this relationship was observed during early studies involving external ion injection into a QITMS.26,28 Figure 3a also indicates that there is a different linear relationship for singly charged and doubly charged ions. The equation for the +1 ions is y ) 0.024x + 33.2, whereas the equation for the +2 ions is y ) 0.045x + 21.7. The width of the Gaussian distribution was also determined for each ion (Figure 3b), but no apparent correlation between m/z and width was found. The widths of the Gaussian distributions were found to be 8 ( 2 Da. The reproducible Gaussian relationship between ion intensity and LMCO during ion accumulation allows the development of an intensity encoding scheme for multiplexed MS/MS. In doing so, a previously considered disadvantage of quadrupole ion traps can be turned into an advantage. The means by which this intensity encoding scheme is applied is illustrated in Figure 4. If a given set of precursor ions is accumulated at one LMCO during the acquisition of a primary spectrum (1°) and then accumulated at a different LMCO during the acquisition of a secondary spectrum (2°), the ions’ intensity changes can be predicted based upon the known Gaussian distributions. If this change is known for all precursor ions, then it will apply to all product ions that are produced from a given precursor ion. The change in precursor ion intensity between a primary and secondary spectrum can predicted by considering the general form of a Gaussian distribution (eq 1).
y)
2 2 1 e-(x-c) /2w wx2π
(1)
e-([LMCO(1°)-c] /2w ) 2
F1°-2° )
e
2
-([LMCO(2°)-c]2/2w2)
(2)
In eq 2, LMCO(1°) is the low-mass cutoff used for ion accumulation in the primary spectrum and LMCO(2°) is the lowmass cutoff used during ion accumulation in the secondary spectrum. The center of the distribution (c) can be determined for each precursor ion using Figure 3a, and width of the Gaussian distribution (w) is assumed to be equal to 8 Da for each precursor ion based on the data in Figure 3b. Application of the Intensity Encoding Scheme to a Mixture of Peptide Ions. The following example illustrates the application of this intensity encoding scheme for five peptide ions. The singly charged ions of TRH-Gly (m/z 421), angiotensinogen fragment 11-14 (m/z 482), leucine enkephalin (m/z 556), β-casomorphin (m/z 659), and leucokinin V (m/z 783) are used. Figure 5a shows the mass spectrum of these five ions acquired at a LMCO of 48. In Figure 5b, the spectrum obtained after isolation of the five ions using SWIFT is shown. Upon simultaneously subjecting all five ions to collision-induced dissociation (CID) under the same accumulation conditions, the product ion spectrum in Figure 5c is generated. We refer to this product ion spectrum as the primary spectrum. This process is then repeated at a LMCO of 58 Da, which produces the spectra in Figure 5d and e. The spectrum in Figure 5e is referred to as the secondary spectrum. It should be noted that in practice the spectra shown in Figure 5b and d are not necessary to the success of this approach because the relationships shown in Figure 3 allow the precursor ion intensity changes to be predicted. Figure 5b and d illustrate how eq 2 can be used to predict the precursor ion intensity changes when the LMCO during ion accumulation is changed. The calculated and observed changes in precursor ion intensities are given in Table 1. With the exception of TRH-Gly, which will be explained later, there is good agreement between the observed and predicted intensity changes. Analytical Chemistry, Vol. 76, No. 24, December 15, 2004
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Figure 6. (a) Ratio spectrum obtained by dividing the primary spectrum by the secondary spectrum. (b) Expanded region of the ratio spectrum from m/z 450 to 625. Table 1. Observed and Calculated Ratios of Precursor Ion Intensities at LMCO of 48 (Primary Spectrum, 1°) and 58 Da (Secondary Spectrum, 2°) ion intensity ratio (1°/2°)
Figure 5. (a) Mass spectrum of a mixture of five peptide ions acquired using a LMCO of 48 during ion accumulation. (b) Isolation of the five precursor ions after accumulation of the ions using a LMCO of 48. (c) Dissociation of the five precursor ions isolated in b. (d) Isolation of the same five precursor ions after accumulation of the ions using a LMCO of 58. (e) Dissociation of the five precursor ions isolated in d. 7350 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004
peptide ion
observed
calculated
leucokinin V (m/z 783) β-casomorphin (m/z 659) leucine enkephalin (m/z 556) angiotensinogen (m/z 482) TRH-Gly (m/z 421)
1.2 2.1 2.4 3.2 1.9
1.2 1.9 2.7 3.7 4.5
While some differences in the product ion spectra shown in Figure 5c and e are apparent, these spectra are too complex to simply visually identify the intensity changes of the product ions. A more convenient means of determining the intensity changes and thus identifying the precursor ion from which each product ion arises is to use a ratio spectrum. The ratio spectrum (Figure 6a) is obtained by dividing the primary product ion spectrum by the secondary product ion spectrum. To minimize any noise that would ultimately be magnified in the ratio spectrum, product ions with intensities below 115, which is 10% of the most abundant peak in the secondary spectrum, have been subtracted from both the primary and secondary spectra before dividing the two spectra. Although this certainly removes some useful information, this is a necessary step to obtain a more readily interpretable ratio spectrum. The product ions arising from each precursor ion can then be determined using the ratio spectrum. Finding the correct precursor ion/product ion linkages is done by identifying peaks in the ratio spectrum that correspond to the observed precursor ion changes in Table 1. As an illustration of how this is accomplished, Figure 6b shows an expanded region from m/z 450 to 625, which shows 10 different product ions. The precursor ions identified for each of these product ions are given in Table
Figure 7. Full product ion spectrum of leucokinin V with peak identification. The peaks labeled with a (*) correspond to correctly assigned product ions, those labeled with a (+) correspond to product ions with m/z ratios within 5 Da of one of the precursor ions, and the one labeled with a (?) corresponds to an incorrectly assigned ion. Table 2. Product Ions Observed in the Ratio Spectrum in the m/z Range of 450-625
product ion (m/z)
intensity ratio (1°/2°) of product ion
465 487 493 495 500 505 526 538 597 614
2.3 1.2 2.4 2.1 2.2 1.2 1.7 2.7 2.0 2.0
identified precursor ion from which the product ion arises (precursor ion intensity ratio from Table 1) leucine enkephalin (2.4) leucokinin V (1.2) leucine enkephalin (2.4) β-casomorphin (2.1) β-casomorphin (2.1) leucokinin v (1.2) leucine enkephalin (2.4) β-casomorphin (2.1) β-casomorphin (2.1)
2. The observed intensity ratio changes allowed all the product ions in this region to be correctly identified with the exception of the product ion at m/z 526. A product ion was linked to a particular precursor ion if its intensity ratio was within 10% of the precursor ion’s intensity ratio. For example, both the product ions at m/z 487 and 505 were found to have an intensity ratio change of 1.2, which is the same intensity ratio change for the parent ion leucokinin V. Consequently, these two product ions can be assigned to leucokinin V. Similarly, the product ions at m/z 495, 500, 597, and 614 have intensity ratio changes of 2.1, 2.2, 2.0, and 2.0, respectively. These values are all within 10% of 2.1, which is the intensity ratio change with which β-casomorphin was encoded. The product ion at m/z 526, which was not correctly linked to its precursor ion, has an observed change of 1.7 whereas its precursor ion, leucokinin V, has an intensity change of 1.2. In this case, the likely explanation for the misencoding is poor ion statistics because this product ion has an intensity that is very low (155 in the secondary spectrum). Another means by which the data can be displayed is to show the normal product ion spectrum of one of the precursor ions with an indication of the product ions that were identified during the multiplexed MS/MS analysis. Figure 7 shows the product ion spectrum for leucokinin V and indicates the peaks that were correctly identified during the multiplexing experiments. Peaks marked with an (*) are product ions that were correctly assigned to have come from leucokinin V during the multiplexing experiment. From Figure 7, it is clear, though, that some information
has been lost in using the multiplexing approach. The information that is lost falls into four categories. The first category corresponds to the product ions that were not observed in the ratio spectrum, and these product ions are unlabeled in Figure 7. These product ions were not identified in the ratio spectrum because they had intensities that fell below the arbitrary intensity threshold that was used to minimize any noise introduced upon dividing the primary and secondary spectra. The second category of product ions that are unassigned corresponds to product ions that have m/z values close to the m/z values of one of the five precursor ions. The product ions in this category are labeled with a (+). Because, the dissociation waveforms were applied with a width of 10 m/z units for each precursor ion, the product ions at m/z 418, 422, and 655 from protonated leucokinin V were likely further dissociated because they had m/z values close to the precursors TRH-GLY (m/z 421) and β-casomorphin (m/z 659). Upon formation, these product ions were resonantly excited because they have secular frequencies within one of the frequency ranges applied during the dissociation waveform used to perform CID on the five precursor ions. The third category of product ions corresponds to product ions that do not have intensity ratios close to the precursor ion, and these product ions are labeled with a (?). For leucokinin V, m/z 526 falls into this category. As mentioned above, the intensity ratio of this product ion is 1.7, while the precursor ion, leucokinin V, has an intensity ratio of 1.2. In all cases, a 10% error was arbitrarily placed on the intensity ratios of product ions observed in the ratio spectrum. If a product ion’s intensity ratio was not within 10% of the intensity ratio of a precursor ion, it was considered unidentified. A fourth category of unassigned product ions is described by those ions that have the same m/z ratio as a product ion from another precursor ion. In these cases, the product ion intensity ratio would not be expected to correspond to either precursor ion. No product ions from leucokinin V fall into this category. All the results obtained from the ratio spectrum (Figure 6a) are summarized in Table 3. This table indicates the percentage of product ions identified for each of the five precursor ions. The number of product ions for each precursor ion was determined by identifying the 14 most intense product ions above 5% relative intensity from the product ion spectrum of the precursor ion by normal MS/MS. Of the 50 product ions arising from CID of the 5 precursor ions, 33 of them were correctly identified. The remaining 17 product ions that were not identified fall into one of the four categories mentioned above. The unidentified product ions are ions that did not appear in the secondary spectrum above the noise reduction threshold. A total of five product ions fall into this category. The SWIFT ejected ions refer to product ions that had m/z values close to the m/z of a precursor ion, so the SWIFT dissociation waveform caused these ions to be further dissociated. A total of five product ions fall into this category. Coincident ions indicate product ions that have the same m/z as a product ion from a different precursor, so the intensity ratio is expected to be incorrect. A total of four product ions fall into this category. In theory, a third product ion spectrum (or tertiary spectrum) could be taken in which the precursor ion intensities are changed by a different ratio, and these coincident ions could then be correctly linked to the appropriate precursor ion. If it is assumed that coincident product ions arise from at most two precursor ions, Analytical Chemistry, Vol. 76, No. 24, December 15, 2004
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Table 3. Results for the Product Ions from the Five Precursor Ions Used To Illustrate the Multiplexing MS/MS Approach
peptide ion
no. of product ions
product ions correctly identified
% correctly identified
ions identified incorrectlya
unidentified ionsb
coincident ionsc
SWIFT ejected ionsd
leucokinin casomorphin leucine enkephalin angiotensinogen TRH-Gly
14 10 14 8 4
10 8 10 2 3
71 80 71 25 75
1 0 0 2 0
0 0 1 4 0
0 1 2 0 1
3 1 1 0 0
a Product ions with intensity ratios that do not correspond to any precursor ion intensity ratio. b Product ions that did not appear above the noise reduction threshold. c Product ions formed by more than one precursor ion. d Product ions with m/z ratios within 5 Da of one of the precursor ions.
then simple matrix algebra could be used to deconvolute the data. The incorrectly identified product ions in Table 3 are those having intensity ratios that do not correspond to the intensity ratio of any precursor ion. A total of three product ions fall into this category. In these three cases, the likely cause of the incorrect encoding can be attributed to the low intensity of these product ions, which leads to relatively poor ion statistics. These three ions, m/z 235 and 252 from angiotensinogen fragment 11-14 and m/z 526 from leucokinin V, have intensities of 139, 140, and 155, respectively, which is close to the chosen noise threshold of 115. This result indicates that while careful choice of the noise threshold needs to be made to avoid the loss of too much data, the level chosen for the noise threshold will also impact the number of product ions that are incorrectly identified. With this multiplexed method, 66% of product ions were identified successfully. It is notable from Table 3 that a relatively low percentage of the product ions from angiotensinogen fragment 11-14 were identified. This result is due to the low intensity of the precursor ion (Figure 5a, b, and d), which leads to 75% of the product ions having intensities either near or below the arbitrarily defined noise threshold. If the results from angiotensinogen fragment 11-14 are not taken into account, then 74% of the product ions overall are identified correctly. Only 6% of the product ions in total were misidentified after data processing. Improper Encoding of TRH-Gly. As indicated earlier (Table 1), the peptide TRH-Gly (m/z 421) did not exhibit the predicted intensity ratio change. The reason for this can be understood from Figure 8. The vertical lines in Figure 8 indicate the LMCO values at which ions were accumulated to generate the primary and secondary spectra. At both of these LMCOs, it is clear that the intensity of the TRH-Gly ions are not actually on the Gaussian distribution but rather on a “tail” of the distribution. This tailing is present to some extent in all ion intensity distributions, although the tailing is most severe for lower m/z ions. This deviation from a Gaussian distribution only has a major detrimental effect on predicting the intensity change if the LMCO values used to acquire the primary and secondary spectra occur in this “tailing” region of an ion’s distribution. Thus, in the example shown here, the encoding of TRH-Gly is most dramatically impacted by this effect, although the deviations between the predicted and observed intensity ratios for some of the other precursor ions may also be attributed to a departure from a pure Gaussian distribution. As this deviation occurs to some extent in all ion distributions, further work is required to clarify these “modified” Gaussian distributions to better ensure proper intensity encoding. 7352 Analytical Chemistry, Vol. 76, No. 24, December 15, 2004
Figure 8. Ion intensity of the (M + H)+ species of TRH-Gly as a function of the LMCO used during ion accumulation. The solid line indicates the LMCO value used during acquisition of the primary spectrum, and the dotted line indicates the LMCO value used during acquisition of the secondary spectrum.
Future Prospects. The work presented here has demonstrated a way of performing multiplexed MS/MS experiments on a quadrupole ion trap mass spectrometer. There are several aspects of this approach, however, that need to be addressed in the immediate future to further optimize this method. First, studies are required to better understand the relationship between ion intensity and LMCO during ion accumulation. In this work, we have assumed a Gaussian distribution, but data like that shown in Figure 8 clearly indicate that the distributions may not be best represented by a single, simple Gaussian distribution. Second, the effects of ion injection parameters and helium pressure on both the center of Gaussian distributions and widths of these distributions need to be investigated. Studying how ion injection parameters and helium pressure impact the ion intensity distributions as a function of LMCO will allow these distributions to be better understood and perhaps controlled. A third aspect that needs to be addressed is the relatively narrow m/z range of precursor ions that can be effectively multiplexed in a given experiment. Two factors currently limit the m/z range. The first relates to the relatively narrow distribution widths that are evident in Figures 2, 4, and 8, which limit the range of precursor ions that can be effectively accumulated at given LMCO values. More careful study of the ion injection parameters may allow these distributions to be broadened. This limitation could also be addressed by acquiring two sets of primary and secondary spectra, where the first set would be for a lower m/z range of precursor ions and the second set would be for a
higher m/z range of precursor ions. For more complex mixtures than shown here, this approach could be very useful. And while the second set of primary and secondary spectra would add time to the overall analysis, this approach would still provide time savings and would still maintain the multiplexing advantage for a relatively large collection of precursor ions (N > 6). A second limiting factor on the m/z range is the low CID efficiency of precursor ions that are stored at low qz values (qz < 0.2) during resonance excitation. As a broader m/z range of ions is studied, the higher m/z precursor ions will necessarily be stored at lower qz values during resonance excitation to ensure that the lower m/z precursor ions are stored at qz values that provide sufficient product ion dynamic range. Several means of increasing CID efficiency at low qz values have been discussed in the literature, and these include the addition of heavier bath gases into the vacuum system29,30 and the use of infrared multiphoton dissociation (IRMPD).31-34 Alternatively, the multiplexing experiment could be done in two steps, first on a low m/z range of precursor ions and then on a higher m/z range of ions as alluded to in the previous paragraph. CONCLUSIONS In this work, we have demonstrated a multiplexed MS/MS method on a quadrupole ion trap mass spectrometer. This method offers the potential advantages of shorter analysis times, higher sample throughput, and better signal-to-noise ratios than conven(29) Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1996, 68, 463-472. (30) Vachet, R. W.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1996, 7, 11941202. (31) Stephenson, J. L.; Booth, M. M.; Shalosky, J. A.; Eyler, J. R.; Yost, R. A. J. Am. Soc. Mass Spectrom. 1994, 5, 886-893. (32) Colorado, A.; Shen, J. X.; Vartanian, V. H.; Brodbelt, J. Anal. Chem. 1996, 68, 4033-4043. (33) Goolsby, B. J.; Brodbelt, J. S. Anal. Chem. 2001, 73, 1270-1276. (34) Payne, A. H.; Glish, G. L. Anal. Chem. 2001, 73, 3542-3548.
tional MS/MS methods due to its multiplexing nature. The product ion spectra of N precursor ions can be gathered in as little as two product ion spectra. In other words, the same quality MS/MS data obtained normally for N precursor ions can be obtained in a fraction (i.e., 2/N) of the time. This approach is likely to be advantageous for online analyses with LC as coelution of components could be addressed by this multiplexing method instead of using peak parking methods. Relative to peak parking methods, this approach would increase MS sensitivity and sample throughput and maintain the resolution of the LC separation. The multiplexing method in itself is generally fairly simple as only two arbitrary waveforms are required for the duration of the experiment, one for isolation and one for dissociation. The need for only two waveforms stems from ions being intensity encoded by the inherent mass bias present during ion accumulation. Two current limitations are evident with this technique. First, relatively narrow widths of the ion intensity distributions as a function of accumulation voltage exist, which limits the range of precursor ions that can be studied simultaneously. This limitation can perhaps be overcome by modifying ion injection parameters, helium pressure, or both. A second limitation is poor MS/MS efficiencies of ions stored at low qz values during resonance excitation, which also limits the effective m/z range of this method. This limitation can be addressed by using either IRMPD or adding heavier gases to the vacuum system during CID. ACKNOWLEDGMENT The authors thank Dr. Ryan Danell from the Rowland Institute at Harvard University for his helpful discussions concerning the application of SWIFT in these experiments. Received for review July 16, 2004. Accepted October 1, 2004. AC048955D
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