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Active Chemical Background and Noise Reduction in Capillary Electrophoresis/ Ion Trap Mass Spectrometry Roswitha S. Ramsey, Douglas E. Goeringer, and Scott A. McLuckey' Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 -6365
INTRODUCTION Coupling mass spectrometry with condensed-phase separation techniques, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), has been facilitated by electrospray ionization because it is effected directly on solutions containing the analyte of interest. The potential for electrospray as an interface for on-line condensed-phase separations coupled with mass spectrometry was recognized early in the modem development of the technique.' HPLC and CE techniques have since been coupled with a variety of mass spectrometers using several electrospray interface variations.2-17 Recently, attention has focused on the relative merits of the quadrupole ion trap as the mass spectrometer coupled with HPLCleJQ and with CE.20~21We have recently interfaced CE with a quadrupole ion trap modified to accommodate electrospray22and focused our attention initially on the background and chemical noise that contribute to the total ion current in the electropherogram. Clearly it is desirable to observe signals due to analyte species in the total ion current (TIC) electropherogram for (1)Whitehouse,C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1986,57,675. (2)Smith, R. D.; Udseth, H. R.; Barinaga, C. J.; Edmonds, C. G. J. Chrornatogr. 1991,559,197. (3)Wahl, J. H.; Goodlett, D. R.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1992,64,3194. (4) Olivares, J. A.;Nguyen, N. T.; Yonker, C. R.; Smith, R.D. Anal. Chem. 1987,59,1230. (5)Smith, R.D.; Olivares, J. A,;Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988,60,436. (6)Smith, R.D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988,60, 1948. (7)Loo,J. A.;Jones,H. K.;Udseth,H. R.; Smith, R. D. J.Microcolumn Sep. 1989,1,233. (8) HWK, E. C.: Wachs, T.: Conboy, J. J.; Henion, J. D. Anal. Chem. 1990;62,718A. (9)Hopfgartner,G.; Wachs,T.;Bean, K.;Henion, J.Ana1. Chem. 1992,
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(10)Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987,59, 2642. (11)Lee, E.D.;Muck, W.; Covey, T. R.; Henion, J. D. Biomed. Enuiron. Mass Spectrom. 1989,18,844. (12)Huang, E.C.; Henion, J. D. J. Am. SOC.Mass Spectrom. 1990,1, 158. (13)Thibault, P.; Paris, C.; Pleasance, S. Rapid Commun. Mass Spectrom. lWl,5,484. (14)Thibault, P.; Locke, S.;Paris, C.; Pleasance, S.Proceedings of the 40th Conference on Mase Spectrometry and Allied Topics, Washington, DC, May 31, 1992;p 1953. (15)Goodlett, D. R.; Wahl, J. H.; Udseth, H. R.; Smith, R. D. J. Microcolumn Sep. 1993,5,57. (16)Moseley, M. A.;Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K. B. J.Am. SOC.Mass Spectrom. 1992,3, 289. (17)Niessen,W.M.A.;Tjaden,U.R.;vanderGreef,J. J.Chromatogr. 1993,636,3. (18)McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63, 375. (19)Van Berkel, G. J.; Ramsey, R. S.;McLuckey, S.A.;Glish, G. L. Proceedings of the 40th Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31,1992;p 711. (20)Schwartz,J. C.; Jardine, I. Proceedings of the 40th Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31,1992; p 707. (21)Mordehai, A.; Henion, J. Proceedings of the 40th Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31,1992; p 197. (22)Van Berkel, G. J.; Glish,G. L.; McLuckey, S. A.Anal. Chem. 1990, 62,1284. 0003-2700/Q3/0365-3521$04.00/0
detection purposes. Frequently, analyte signals are not apparent in the TIC electropherogram but are clearlyobserved in the extracted ion current profile.18 The analyte quantities at which analyte signals disappear into the TIC background depend both upon the magnitude of the analyte signal and upon the background levels. The background level can depend upon a variety of factors including CE buffer composition, pH, flow rates, interface conditions, etc.6 Approaches to affect background signals in electrospray CE/ MS have included, for example, variation of flow rate,l6 use of volatile buffers, column treatment, and adjustment of pH and sheath conditions.13J6J7 In order to observe peaks in the TIC electropherogram, regardless of the CE and electrospray conditions, the mass spectrometer must in some way discriminate against the background. It is important to recognize that electrospray produces current continuously and that the total ion signal arising from electrospray may or may not change when analyte is present. If the background ions fall a t mass/charge values significantlydistant from those derived from the analyte, the mass spectrometer can simply avoid them by not scanning over those regions. However, the background ions can pose problems when they fall in mass/ charge regions in which analyte species might be expected. It is the background signals that appear in the mass/charge window analyzed by the mass spectrometer, therfore, that largely determine the point at which analyte is no longer observable in the TIC electropherogram. For unknowns, it is desirable to maintain as wide a mass/charge window as possible to minimize the likelihood for missing a mixture component. There are, of course, several approaches for manipulating the datato make analyte signalemore apparent. These include, for example, background subtraction and plotting the base peak signal as opposed to the total ion signal.M These are often quite useful approaches, although the background signal can be highly variable from one scan to the next. In any case, it would be desirable to physically remove those species that contribute to the background to minimize detection limits in the TIC electropherogram. We report here a combination of broad-band collisionalactivation and resonance ejection that we have found to be useful in improving signal/background and signal/noise ratios in TIC electropherograms obtained with CE coupled with a quadrupole ion trap.
EXPERIMENTAL SECTION Experiments were performed with a Finnigan (San Jose, CA) ion trap m w spectrometer ( W S )modified to allow for injection of ions from external ion sources.22 The CE apparatus was constructed in our laboratoryand conaiats of a Hypotronics highvoltage power supply (Brewster, NY), a safety interlock box, and an Isco Model 5500 UV absorbance detector (Lincoln, NE). The cathode (lowvoltage) end of the CE column was directedthrough a 21-gauge, dome-tipped, stainless steel needle through which a liquid sheath was also transported. The liquid sheath was comprised of 75% acetonitrile, 24% water, and 1%acetic acid and was delivered at a rate of 1 pL/min by a syringe pump (Harvard Apparatus, Inc., Cambridge, MA). The needle was maintained at +3 to +4 kV to produce the electrospray at the capillary terminus and to provide the negative bias relative to the anode (injection end of the capillary) to drive the electrophoresis. A potential of +20 kV was used for separations, and @ 1993 Amrlcan Chemlcal Society
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Ilh Flgurr 1. (a) Normailred mass spectrum of trls-phosphate buffer mixed with electrospray sheath. (b) Mass spectrum of (a) with the vertical scale expanded by a factor of 16.
all injections were made electrokinetically at 10kV. Separations were performed on a 56-cm-long,50-pm-i.d., fused-silicacolumn (Polymicro Technologies, Phoenix, AZ) using 75 mM trisphosphate buffer at pH 7.6. The peptides were obtained from Sigma (St. Louis, MO). Noise signals were applied to the ion trap end caps during the ion injection period, which extended to as much as 400 ms. A description of the electronics and experimental setup used to couple the noise to the ion trap has been reported.29 The amplitude of the noise was adjusted to maximize signal/noise ratios for the analyte ions. Ten-millisecond single-frequency resonance ejection signals of 6 V (p-p) each were used just prior to the mass analysis scan to remove the major buffer-related ions. Resonance ejection was used in all experiments to extend the mass/charge range of the ITMS to 1300.
RESULTS AND DISCUSSION In addressing the issue of background signals with the CEI ITMS system, we noted that there were two types of species that contribute to the chemicalbackground. One type consists of ions that consistently appear in the mass spectrum a t discrete m / t values. These ions are usually related to the buffer. The other type of chemical background tends to give background signalsat random locations in the mass spectrum from one scan to the next. Evidence suggests (see below) that these signals arise largely from the decomposition of loosely bound clusters that occur during themass scan. Figure 1illustrates the two types of background species with a mass spectrum acquired under conditions of "moderate" background. Figure l a shows the normalized mass spectrum and indicates that the dominant species in the spectrum consists of protonated tris(hydroxymethy1)aminomethane (tris) and the proton-bound dimer of tris. Figure l b shows the spectrum with the vertical scale expanded by a factor of 16. Signals due to the protonated trimer and protonated tetramer of tris are now clearly apparent, as are a number of peaks that do (23) McLuckey, S. A.; Goeringer, D. E.; Glish, G. L. Anal. Chem. 1992,
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not appear consistently from one scan to the next. Although these ions tend to be, at most, a few percent of the intensity of the base peak, their large numbers make up a significant fraction of the total ion signal in the spectrum. The nature of the background species giving rise to the random signals in the spectrum was revealed when it was noted that delay times of several hundreds of milliseconds after ion injection and prior to mass analysis resulted in a noticeable reduction of the random signals and an increase in the signal due to protonated tris and the protonated tris dimer. We have previously studied the decompositions of protonated water and protonated methanol clusters in the ion trap% and have seen that cluster decomposition on the tens to hundreds of milliseconds time frame can occur for species with the appropriate kinetic stabilities. In previous work in coupling HPLC with the ion trap we also noted a reduction in the chemical background when we employed random noise collisional activation to dissociate the analyte ions.lQ It is noteworthy that background levels were not observed to increase with increasing ion accumulation time beyond a few hundred milliseconds whereas analyte ion signals increased linearly with ion accumulation time to a t least 1s. This observation, and the fact that the peak shapes associated with the analyte ions were not observed to be affected significantly by the range of ion accumulation periods used in this work, suggest that the background signalsdo not arise from ion-ion interactions. We therefore conclude that these background signals arise from decomposing cluster ions. If so, a possible approach to removing or reducing the chemical background that appears a t random in the mass spectrum would be to accelerate the decomposition of loosely bound species by applying a relatively low amplitude noise signal during or after ion injection to collisionally heat all of the ions. If analyte ion decomposition is not desired, the amplitude must be selected so that covalently bound species are not collisionally heated sufficiently to fragment. Therefore, the application of noise discriminates against the background ions on the basis of ion stability. Many of the cluster ions are buffer-related and they eventually decompose to the most stable buffer-related ions, protonated tris and the proton-bound dimer of tris. In order to reduce the background in the electropherogram due to the stable bufferrelated ions, single-frequency resonance ejection is used to eject them prior to the mass scan. It is important that the resonant ejection periods occur after the noise period since decomposition of some of the cluster ions forms the stable buffer-related ions while others yield products that fall below the low-mass cutoff of the ion trap. It is also important to realize that the background ions removed by resonance ejection are being discriminated against on the basis of mass/ charge so that any analyte ions that might fall at this mass/ charge are also lost. The reconance ejection periods create blind spots in the mass spectrum whereas the noise period does not. In all of the data shown here, the maselcharge range covered by the ion trap is 100-1300 with uholes"at m/z 122 (protonated tris) and 243 (protonated tris dimer). (Note that analyte ions that might appear at these masslcharge values in the absence of resonance ejection would probably be obscured by the much more abundant buffer-related ions. Therefore, the use of resonance ejection to eject the bufferrelated ions would seem to have little negative impact on the analysis.) A variety of factors can play a role in the background level. These include, for example, sheath composition, buffer composition, flow rates, vacuum-atmosphere interface conditions, etc. We have not fully characterized all of these (24) McLuckey, S. A.; Asano, K. G.; Glish, G. L.; Bartmess, J. E. Int. J. Mass Spectrom. Ion Processes 1991, 109, 171.
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Flaurr 2. Total ion electropherograms of a mixtures of bradyklnin (B, 320 fmol), xenopsin (X, 370 fmoi), and neurotensin (N, 440 fmoi) acqulred using (a) no means for background reduction, (b) resonance ejection to remove ions at mlz 122 and 243, (c) low-level broad-band coiiisionai activation without resonance ejection, and (d) both resonance ejection and broad-band collisional activation.
parameters for their effects on background signals nor have we identified all of the variables. We find, for example, that background levels can vary by as much as 1order of magnitude over the course of a few weeks with a nominally fixed set of conditions. In some cases, it may not be feasible to find the conditions for minimum background levels without significantly affecting the separation or analyte signal levels. The tactica presented here provide means for reducing background independent of the electrospray and separations conditions. Figure 2 illustrates the effect of various combinationsof noise and reaonance ejection on the appearance of an electropherogram under the conditions used to acquire the background mass spectra shown in Figure 1,which represents a moderate background level. In each case, a mixture of bradykinin (320 fmol), xenopsin (370 fmol), and neurotensin (440 fmol) was injected onto the column. No measures were taken to discriminate against background ions for the electropherogram of Figure 2a. The signal due to neurotensin is apparent but not those for bradykinin and xenopsin. All three mixture components were clearly apparent, however, in the extracted ion profilesfor their respectivebase peaks (notshown). Figure 2b shows the electropherogram obtained when protonated tris and the protonated tris dimer were ejected from the ion trap prior to mass analysis,and Figure 2c showsthat obtained when noise was used during injectionbut no resonance ejection of the stable buffer ions was employed. Both show some improvement in signal/background. However, the most dramatic improvement is shown in Figure 3d, where both tactics for background ion diminution were employed. The benefit derived from the use of these measures to reduce background is, of course, highly dependent upon the background level. We have noted reductions in background and peak-to-peak noise levels by factors 20-30 in cases where background levels are high. More modest improvement, like that illustrated in Figure 3, is obtained in cases where the
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3% 6.32 ?ae 9.49 3ee 12.59 4aem llnr Flgure 3. Total ion electropherograms of a mixture of bradykinin (B, 180 fmol), xenopsin (X, 200 fmol), and neurotensin (N, 220 fmoi) under conditions of reiatlvely low background (a) without active background reduction measures and (b) with combined broad-band colilslonai acthratbn and resonance ejection. background level is already relatively low. Figure 3 shows the total ion electropherogramfor the mixture discussed above with injections of 180, 200, and 220 fmol of bradykinin, xenopsin, and neurotensin, respectively. The improvement in signal/noise ratios here is on the order of a factor of 3. From the data it would appear that detection limits in the
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mentioned above might provide even further improvement. It is important to recognize that, since the use of noise for background signal reduction relies on the premise that the background is more readily fragmented than the analyte, particularly fragile analyte ions might also dissociate. Figure 4 shows the averaged, background-subtracted mass spectra of the mixture componentsderived from the electropherogram of Figure 3b. Excellent signal/noise ratios are observed for all three components and all show intense doubly or singly protonated molecules. In fact, bradykinin and neurotensin both show the doubly protonated molecules almost exclusively. Significant fragmentation, however, is reflected in the xenopsin mass spectrum, some of which arises from the application of noise during ion injection. This example illustrates the potential trade-off that could arise from the analysis of readily dissociable analyte species in using noise to discriminate against background species.
(M+2H)'+ M.= 1060
CONCLUSIONS
M,=1673
Figure 4. Averaged and backgroundsubtractedmass spectra of the
mixture components (a)bradyklnln, (b) xenopsin, and (c) neurotensln as they appear in the total ion electropherogramof Figure 3b. Several major fragment Ions observed In (b)are labeledand were also observed in the MS/MS spectrum of doubly protonated xenopsin.
total ion electropherogram might extend down to 10 fmol or so. The use of data manipulation techniques such as those
The combined use of broad-band collisional activation (CAI and one or more subsequent resonance ejection periods can lead to significant reduction in chemical background and chemical noise in the total ion electropherogram acquired from capillary electrophoresis combined with electrospray and ion trap mass spectrometry. Broad-band CA, at the appropriate power level, can discriminate against loosely bound speciessuch as solvent and buffer-derived ions without significantlyaffecting signals due to covalently-boundanalyte ions. Resonance ejection can be used to notch out stable background ions, thereby creating one or more blind spots in the mass spectrum. This combined approach allows for the acquisition of mass spectra over a wide mass/charge range with improved ability to observe analyte peaks followinglowlevel injections.
ACKNOWLEDGMENT This research was supported by the National Institutes of Health under Grant GM45372. Dr. Gary J. Van Berkel is acknowledged for helpful discussions. Oak Ridge National Laboratory is managed for the U.S.Department of Energy by Martin Marietta Energy Systems, Inc. under Contract DE-AC05-840R21400. RECEIVED for review June 14, 1993. Accepted September 14, 1993.
