Detection of Fast Capillary Electrophoresis Peptide and Protein

Anal. Chem. 1996, 68, 1480-1485

Detection of Fast Capillary Electrophoresis Peptide and Protein Separations Using Electrospray Ionization With a Time-of-Flight Mass Spectrometer J. Fred Banks, Jr.* and Thomas Dresch

Analytica of Branford, 29 Business Park Drive, Branford, Connecticut 06405

Fast capillary electrophoresis (CE) separations on bare and coated fused silica have been detected on-line using a time-of-flight mass spectrometer which is capable of acquiring spectra at a rate suitable for the narrow peaks generated by CE. Protein and peptide separations producing peaks 1-2 s in width have been detected with an integration time of 0.125 s/spectrum, each spectrum being the sum of 1024 complete m/z range scans. The effects of the liquid sheath flow rate, electric field strength, and integration time on sensitivity and peak shape have been examined. A sensitivity limit of 8 fmol has been established for leucine enkephalin. Since its introduction, modern capillary electrophoresis1 (CE) has rapidly matured into a useful and accepted analytical technique. The growing interest in this separation method stems from the rather high separation efficiencies potentially achievable (107 theoretical plates) and the sensitivity limits demonstrated when using advanced detection schemes. Because of the small sample volumes required (a few nanoliters or less), CE is quickly becoming the analytical tool of choice for applications which are truly sample limited. Mass spectrometry (MS), when combined with electrospray (ES) ionization, is an advanced detection technique which can provide molecular weight information as well as structural clues from fragmentation, which can be induced either inside the ES source itself (in-source fragmentation)2-4 or through the use of multiple stages of mass analysis.5,6 ES-MS is thus a likely detector for CE separations, especially in the case of biological applications and drug metabolites, where the absolute limit of separation efficiency and quality or quantity of detector information is often required. The subject of CE-MS in general has been described in a number of recent reviews.7-10 (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298. (2) Voyksner, R. D.; Pack, T. Rapid Commun. Mass Spectrom. 1991, 5, 263. (3) Banks, J. F.; Shen, S.; Whitehouse, C. M.; Fenn, J. B. Anal. Chem. 1994, 66, 406. (4) Starrett, A. M.; Didonato, G. C. Rapid Commun. Mass Spectrom. 1993, 7, 7. (5) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993, 65, 425. (6) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1991, 3, 60. (7) van der Greef, J.; Niessen, W. M. A. Int. J. Mass Spectrom. Ion Processes 1992, 118/119, 857. (8) Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1993, 636, 3. (9) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574.

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Experience has shown that the interfacing of CE and ES is nontrivial and more complicated than LC-ES interfacing in that an electrical contact must be made with the CE column outlet (a glass dielectric material) in order to provide a circuit for (i) the CE current flow and (ii) the electrospray current flow. Several approaches have been designed to solve this difficulty, including the use of (i) a coaxial sheath flow both with11,12 and without13-15 the use of additional nebulizing gas, (ii) a metal-coated, sharpened CE column outlet,16,17 (iii) a gold wire electrode inserted into the CE column outlet,18 and (iv) a liquid-junction interface.19,20 The metal-coated CE column outlet has the advantage that no sheath flow is needed, and thus no sample dilution occurs. It has the disadvantages, however, that ES-compatible buffers must be used with the CE separation and that specialized CE columns must be prepared. The use of an inserted gold wire electrode requires neither sheath flow nor a specialized CE column. It does, however, imply the correct placement of the electrode as well as, again, the use of ES-compatible buffers with the CE separation. Finally, the liquid-junction technique uses an additional buffer reservoir to electrically isolate the dielectric CE column outlet and a conductive metal ES needle. This method requires the careful alignment of the CE column outlet and the ES needle. Because the coaxial sheath flow technique has proven to be simple, reliable, and easy to implement, and because we are most familiar with it due to our previous experience in the coupling of capillary LC and ES, we have focused our efforts on advancing this type of interface. Toward this end, we have developed an improved CE-MS probe21 which incorporates the use of a built-in translation device, allowing the correct positioning of the CE column exit relative to the sheath liquid flow tube exit. In this (10) Cai, J.; Henion, J. J. Chromatogr. 1995, 703, 667. (11) Thibault, P.; Paris, C.; Pleasance, S. Rapid Commun. Mass Spectrom. 1991, 5, 484. (12) Thibault, P.; Pleasance, S.; Laycock, M. V. J. Chromatogr. 1991, 542, 483. (13) Smith, R. D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988, 60, 1948. (14) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1988, 60, 436. (15) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230. (16) Wahl, J. H.; Gale, D. C.; Smith, R. D. J. Chromatogr. 1994, 659, 217. (17) Wahl, J. H.; Smith, R. D. J. Capillary Electrophor. 1994, 1, 62. (18) Fang, L.; Zhang, R.; Williams, E. R.; Zare, R. N. Anal. Chem. 1994, 66, 3696. (19) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. J. Chromatogr. 1988, 458, 313. (20) Lee, E. D.; Muck, W.; Covey, T. R.; Henion, J. D. Biomed. Environ. Mass Spectrom. 1989, 18, 844. (21) Banks, J. F. J. Chromatogr. A 1995, 712, 245. 0003-2700/96/0368-1480$12.00/0

© 1996 American Chemical Society

way, the optimum CE column position can be adjusted without applying torsional stress to the column body, which eventually may break it. This device also allows for the easy insertion and removal of CE columns so that the effects from different column sizes and chemistries may be investigated with increased convenience. Although CE has been interfaced to various types of electrospray mass spectrometers, including quadrupole,13-15,17,19,22,23 magnetic sector,24,25 Fourier transform ion cyclotron resonance (FTICR),26 quadrupole ion storage traps,27 and time-of-flight (TOF) machines,18 none except TOF, due to its inherent fast scanning capabilities, has the potential to take advantage of narrow peaks and fast separations. Ultimately, our particular TOF analyzer can acquire complete scans at a rate of 8192/s, corresponding to m/z scan rates of ∼2.6 × 108 m/z units/s. These scans are summed or grouped together into appropriate time segments, as small or as large as is required by the time domain of the separation process, to generate complete mass spectra. ES-TOFMS has been previously demonstrated by ourselves and others,28-34 and at least one paper,18 in fact, shows CE-ESTOFMS results. To our knowledge, however, the present report is the first that clearly demonstrates the use of CE-ES-TOFMS with fast separations (100-200 s) generating narrow (1-2 s) peaks. It is in this realm of separation speed (and faster) where the benefits of a TOF mass analyzer become evident in relation to the separation process. Figure 1. Schematic of the CE-ES-TOF system.

EXPERIMENTAL SECTION Capillary Electrophoresis. The CE instrument used was an ATI crystal 300 with a four-position sampler. For the bare silica column, a piece of fused silica, 75 µm i.d. × 365 µm o.d. × 1 m, from Polymicro Technologies (Phoenix, AZ) was used. The CE buffer was 0.1% acetic acid in water. For the amine-coated column, a piece of fused silica, 75 µm i.d. × 365 µm o.d. × 66 cm from Polymicro Technologies was used, and the CE buffer was again 0.1% acetic acid in water. The sheath flow, delivered with a Harvard Apparatus Model 11 syringe pump (South Natick, MA), was pure methanol in all cases. The two types of CE columns used were prepared as follows. For the bare column, the fused silica was washed sequentially with acetonitrile for 10 min, 1 M HCl for 10 min, 1 M NaOH for 10 min, pure water for 10 min, and finally the run buffer for 10 min. For the amine-coated column, the fused silica was washed sequentially with 1 M HCl for 10 min, 1 M NaOH for 10 min, the Beckman Instruments (Fullerton, CA) amine regenerating solution for 10 min, and finally the run buffer for 10 min. (22) Perkins, J. R.; Tomer, K. J. Capillary Electrophor. 1994, 1, 231. (23) Moseley, M. A.; Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 1992, 3, 289. (24) Perkins, J. R.; Tomer, K. B. Anal. Chem. 1994, 66, 2835. (25) Tomlinson, A. J.; Benson, L. M.; Naylor, S. J. Capillary Electrophor. 1994, 1, 127. (26) Hofstadler, S. A.; Wahl, J. H.; Bakhtair, R.; Anderson, G. A.; Bruce, J. E.; Smith, R. J. Am. Soc. Mass Spectrom. 1994, 5, 894. (27) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. Biomed. Environ. Mass Spectrom. 1989, 18, 253. (28) Boyle, J. G.; Whitehouse, C. M. Anal. Chem. 1992, 64, 2084. (29) Boyle, J. G.; Whitehouse, C. M. Rapid Commun. Mass Spectrom. 1991, 5, 400. (30) Sin, C. H.; Lee, E. D.; Lee, M. L. Anal. Chem. 1991, 63, 2897. (31) Michael, S. M.; Chien, B. M.; Lubman, D. M. Anal. Chem. 1993, 65, 2614. (32) Coles, J.; Guilhaus, M. Trends Anal. Chem. 1993, 12, 203. (33) Verentchikov, A. N.; Ens, W.; Standing, K. G. Anal. Chem. 1994, 66, 126. (34) Mirgorodskaya, O. A.; Shevchenko, A. A.; Chernushevich, I. V.; Dodonov, A. F.; Miroshnikov, A. I. Anal. Chem. 1994, 66, 99.

Electrospray. The electrospray ionization source was from Analytica of Branford, Inc. (Branford, CT) and is schematically depicted with the TOF system in Figure 1. This system was similar to its original form except for the use of the new CE-ES probe. The sheath flow CE probe was constructed in a three-layer coaxial arrangement, where the CE column exit was the center or first layer. The second layer, or sheath liquid flow needle, was a piece of stainless steel tubing, 0.0160 in. i.d. × 0.0280 in. o.d., and was just large enough to allow passage of the CE fused silica capillary. The third layer, or gas flow needle, was another piece of stainless steel tubing, 0.0325 in. i.d. × 0.0425 in. o.d. A modest gas flow (50 mL/min) through the third layer needle served only to cool the needle and did not affect the electrospray process itself. This gas flow may find additional utility with negative ion ES, where a bath gas of SF6 or O2 is helpful to suppress the occurrence of a corona discharge in the source. Large windows on three sides of the atmospheric region of the source allowed for the visualization of the electrospray process itself and greatly aided in the optimization of instrumental conditions necessary to obtain good results. The voltages in the source were set as follows: the cylinder electrode (Vcyl) at -2500 V, the endplate electrode (Vend) at -3700 V, and the capillary entrance (Vcap) at -4500 V. The use of a dielectric capillary having metal coating on both ends to transfer the ions generated from atmosphere into vacuum is important in this case, as it allows for the CE column exit and ES spray needle to be maintained at ground potential while the source electrode voltages are maintained at high negative potential. ES source designs, which depend on the application of a positive voltage to the spray needle itself and ground potential to the source electrodes, have two disadvantages when CE is used. First, the CE column exit and Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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thus the ES spray needle must be floated at the required potential (several kilovolts), causing the resulting electric field applied to the CE capillary to be the difference between the CE voltage and the spray needle voltage. Second, if the ES source is operational during the injection phase and vial-transfer phases, the sample loaded on the capillary will have a tendency to migrate back out of the capillary entrance and be diluted in the final run buffer before the final voltage can be applied to the run buffer. This phenomenon has the potential to greatly reduce sensitivity, make qantitation difficult, and degrade the separation quality. Time-of-Flight Mass Spectrometer. From the exit of the ion guiding lenses, the ion beam is focused through a 2 mm aperture into the TOF analyzer chamber. The TOF mass spectrometer was developed by Analytica of Branford, Inc., as a mass detector for high-speed liquid chromatography applications35 and was designed as a short linear instrument of the Wiley/ McLaren type.36 The distance from the center of the extraction volume to the detector surface was 400 mm, and the drift energy in the field-free flight tube was 3000 eV for a singly charged ion. No additional postacceleration was applied in the experiments described here. The continuous beam of ions from the ES source enters the first stage of the Wiley/McLaren accelerator orthogonally to the axis of the TOF spectrometer.33,34,37-39 In each scan cycle, a portion of this beam is sent toward the detector by applying a voltage pulse to the repeller electrode. At high cycle repetition rates, this method renders a highly efficient way of adapting a continuous external ion source to the inherently pulsed TOF analyzer. Electrostatic steering deflectors were used to compensate for the effect of the initial orthogonal velocity component. The injection potential that corresponds to the initial perpendicular motion is constant and equal for all ions. Hence, the steering potential is independent of m/z values. The distribution of ion flight times at the end of the flight tube was measured with an ETP Model AF820 secondary electron multiplier (Auburn, MA). A mass resolution of R(fwhm) ) 1000 was determined for leucine enkephalin at m/z ) 556 using a Tektronix Model TDS 540 digital oscilloscope (Wilsonville, OR), operated at 500 MHz. For rapid and continuous analog-to-digital conversion and signal averaging, the output signal from the electrically floating ion detector was capacitively coupled into a Model 9825 integrating transient recorder (ITR) data acquisition device (200 MHz) from Precision Instruments (Knoxville, TN). A preset number of consecutive, digitized TOF spectrometer scan data were added up by the ITR to constitute a mass spectrum. These averaged spectra were then transferred to a hard disk device for storage. During the experiments described here, the scan repetition rate of the TOF spectrometer was fixed at 8192 Hz. Thus, summing of 8192, 4096, 2024, ... scans/spectrum results in the acquisition of 1, 2, 4, ... spectra/s. At this scan rate, the dead (35) Dresch, T.; Gulcicek, E. E.; Banks, J. F.; Whitehouse, C. M.; Fenn, J. B.; Boyle, J. G. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, May 31-June 4, 1993; p 16. (36) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150. (37) Dodonov, A. F.; Chernushevich, I. V.; Laiko, V. V. Proceedings from the 12th International Mass Spectrometry Conference, Amsterdam, 1991. (38) Dawson, J. H. J.; Guilhaus, M. Rapid Commun. Mass Spectrom. 1994, 3, 155. (39) O’Halloran, R. A.; Fluegge, R. A.; Betts, W. L.; Everette, W. L. Report No. ASD-TDR 62-644, prepared under Contract AF 33(616)-8374; The Bendix Corp. Research Laboratories Division: Southfield, MI, 1964.

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Figure 2. Effect of integration time on TICs from a peptide separation (bare silica column) using the TOF instrument.

time of the data acquisition system due to data transfer was 15%, and thus to allow for precise reconstruction of ion chromatograms, a real-time label was attached to the data set of each spectrum. Quadrupole Mass Spectrometer. The quadrupole instrument was a Model HP5989A from Hewlett-Packard (Palo Alto, CA). It was operated in scan mode with a speed of ∼1000 m/z units/s, allowing one data point for every 0.1 m/z unit to be recorded. The integration time for the detector was set to 100 µs, and a high-energy conversion dynode was used in conjunction with the electron multiplier. Chemicals. Buffers were prepared from distilled water obtained from a Barnstead NANOpure II (Boston, MA) system. Acetic acid, ULTREX II Ultrapure Reagent Grade, was purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Methanol was obtained from Mallinckrodt (Paris, KY). All solvents were filtered through a nylon 66 membrane from the Anspec Co. (Ann Arbor, MI). Peptide and protein samples were purchased from Sigma (St. Louis, MO). RESULTS AND DISCUSSION (A) Peptides Separated on a Bare Fused Silica CE Column. Effect of Integration Time. To determine the required MS integration time, a series of total ion currents (TICs) were acquired for a separation of standard peptides as the integration time was changed. In this case, “integration time” refers to the arbitrary grouping of scans recorded, since these scans are being acquired continually at the nominal rate of 8192 Hz and can be grouped, or more accurately summed, in data registers for nearly any arbitrary length of time. Figure 2 shows the result observed in the TIC for the TOFMS detection of a CE separation of Arg-Gly-Asp, mophiceptin, Val-Pro-Leu, leucine enkephalin, and methionine enkephalin (250 fmol/component) as the integration time was varied. A sheath flow of 4 µL/min of pure methanol was applied. The data taken at the slowest rate used, 4 s/spectrum, are severely distorted, displaying artificially broadened peaks. As the integration time was reduced, decreasing the number of scans summed together to produce the final recorded spectrum, the TICs began to accurately reflect the characteristics of the actual separation. Not until a rate of 0.50 s/spectrum (i.e., 2 spectra/s) did the sampling seem appropriate. It should be noted at this point that the data at 0.5 s/spectrum were recorded at a rate 4-fold faster than the minimum scan time achievable with a quadrupole instrument (2 s/spectrum) for the mass range covered.

Figure 4. Effect of sheath flow rate on ion signal strength from leucine enkephalin peak in TIC from a peptide separation. Figure 3. Comparison of the TICs from a peptide separation (bare silica column) using the TOF and quadrupole instruments.

Comparison of Quadrupole and TOF Analyzers. The data obtained with the TOF instrument (0.25 s/spectrum) are directly compared with the best data possible obtained on the quadrupole instrument (2 s/spectrum) in Figure 3. Even though the CE separation was obviously discernible with the quadrupole analyzer, the TIC from the TOF analyzer is certainly more accurate in depicting the actual separation. The peaks detected from the quadrupole instrument appear wider, are poorly shaped, and show m/z signal strength discrimination in time. This is most evident in the leucine enkephalin peak, which appears to be much smaller, relatively, in the quadrupole TIC. This result may be due to the fact that the apex of the peak is easily missed by the quadrupole MS as it slowly scans the m/z range. As the apex of this peak was migrating past the column exit, the mass analyzer was examining a part of the m/z regime unrelated to this peak. Effect of Sheath Flow Rate. While some chemical effects on the sheath flow liquid40 and CE buffer17,23 have been studied, most physical effects have not been investigated. The most significant physical aspect to our work seemed to be the flow rate of the sheath flow solution. Pure methanol was chosen because its lower surface tension allows for a more stable spray when using sheath flow and because its evaporation rate is higher compared to that of water/methanol mixtures. Also, since the observed CE current was modest, 3.5 µA on average, the need for a conductive modifier in the sheath liquid, such as acetic acid, was eliminated. CE buffers with higher concentrations of salts and salts with higher conductivity would no doubt force the use of a conductive modifier in the sheath liquid. The sensitivity advantages of lowering the conductivity of the CE effluent/sheath liquid have been previously discussed by Smith et al.9 We have also seen that the absence of a modifier, namely acetic acid, in the sheath liquid reduced substantially the background noise in the TIC by decreasing the intensity of the m/z peaks associated with acetic acid ions and the m/z peaks associated with contaminants contained in the acetic acid reagent. The addition of a sheath liquid was expected to reduce the MS signal intensity due to solute zone dilution. This was studied by measuring the intensity of the leucine enkephalin peak in the separation above while varying the sheath flow rate. Figure 4 shows the results obtained from this experiment and indicates that, over the range studied, 2-6 µL/min, the signal intensity was (40) Foret, F.; Thompson, T. J.; Vouros, P.; Karger, B. L.; Gebauer, P.; Bocek, P. Anal. Chem. 1994, 66, 4450.

Figure 5. Effect of sheath flow rate on TICs from a peptide separation (bare silica column) using the TOF instrument.

reduced by less than a factor of 2. These results are encouraging, although the actual signal loss compared to a sheath flow rate of zero, or a sheathless configuration, is only speculative. The effect of sheath flow rate on the apparent separation resolution was also investigated. Here, TICs for the same separation were overlaid, again as the sheath flow was varied from 2 to 6 µL/min. These data are shown in Figure 5, where it appears that, as the sheath flow rate was increased, the separation between the peaks improved slightly, as did the peak widths. An explanation for this occurrence can be found by considering the total volume of liquid present in the Taylor cone area, where the CE column flow and sheath liquid flow meet. As the sheath flow increases, this stable volume of liquid is swept more rapidly, and thus the CE column effluent is diluted and transferred at a higher speed toward electrospray filament that eventually breaks into the micrometer-sized droplets due to classic Rayleigh instability. It follows, then, that at higher sheath flow rates there is considerably less time available for zone broadening from diffusion of the analyte into the supporting liquid matrix in the Taylor cone area. Since the greatest improvement occurred between 2 and 4 µL/ min, and since this flow rate produced a very stable spray, 4 µL/ min was chosen as the optimum value. Sensitivity. The last study performed with this set of conditions was a sensitivity and limit of detection analysis. For this work, successive dilutions of leucine enkephalin were prepared and applied to the CE column using the conditions optimized above for the peptide separation. These data are shown in Figure 6 and indicate that the lowest amount of sample that could be detected on the linear portion of the response curve was 8 fmol. Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

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Figure 6. Signal of leucine enkephalin versus amount injected using the TOF instrument. Figure 8. Comparison of the TICs from a peptide separation (aminecoated column) using the TOF and quadrupole instruments.

Figure 7. Effect of CE electric field strength on TICs from a peptide separation (amine-coated column) using the TOF instrument.

This sensitivity can be compared to that obtained by Verentchikov et al.,33 who reported ES-TOFMS spectra from as little as 10 fmol of cytochrome c by direct infusion, and Fang et al.,18 who reported ES-TOFMS spectra from as little as 40 fmol of cytochrome c loaded onto a CE column. Work on increasing the detection limit continues, and we believe that room for substantial improvements exists with the implementation of new and existing technology. (B) Peptides Separated on an Amine-Coated CE Column. Effect of Electric Field Strength. As our goal was to demonstrate CE-MS with decreased overall separation times and peak widths, we next investigated the use of a coated CE column to reduce the peak broadening usually observed on bare silica for peptide and protein separations. For the same reasons, the column length was reduced to 66 cm, the shortest attainable length between the CE injection point and the electrospray tip. Future efforts will focus on further reducing the column length through the design and construction of a more integrated system. Figure 7 shows the TIC from the separation of the same peptide mixture that was described above, using field strengths of -300 and -450 V/cm. The remaining column dimensions were the same as used previously, as was the CE buffer. Upon examining these data, the need for fast scanning capabilities in the mass detector is further demonstrated, as the required integration time was reduced to 0.125 s/spectrum for the peaks in these separations, which are only 1-2 s wide on average. Also, the increase of field strength by 1/3 has the effect of decreasing the peak width and migration times by 1/3, as predicted by the classical theory of electrophoresis. The results suggest that the CE column and buffer are not being overheated at these field strengths and that even higher fields (i.e., shorter columns and/or higher potentials) 1484 Analytical Chemistry, Vol. 68, No. 9, May 1, 1996

may find use in the future to even further decrease the separation times and peak widths. Important also to note here is the reversal of migration order and applied potential as a result of the amine coating. Comparison of Quadrupole with TOF. Figure 8 shows TIC traces from the same peptide separation as above using the TOF instrument with an integration time of 0.125 s/spectrum and the quadrupole instrument with a scan time of 2 s/spectrum, its fastest possible value. The differences now are far more pronounced and the distortion far more severe than seen above with the bare silica column. With the quadrupole, the first three peaks appear to have comigrated, while the morphiceptin peak is missing entirely. The examination of a single scan from this first “peak” shows the presence of all three compounds, giving at least 3 m/z peaks. With no a priori knowledge about the separation or the sample, these m/z data would be very difficult if not impossible to interpret. No peak is observed for morphiceptin since it elutes entirely while the mass spectrometer is slowly scanning unrelated regions of the m/z spectrum. As also seen in Figures 7 and 8, the peak width of these zones is about 2 s, equal to the time required for the quadrupole to acquire only a single scan over the mass range covered. The overall run time is just under 100 s. Finally, Figure 9 shows a single complete spectrum underneath each peak from the TOF data in Figure 8, representing only 0.125 s of data. The absence of any significant chemical background is primarily a result of the elimination of acetic acid in the methanol sheath flow. (C) Proteins Separated on an Amine-Coated CE Column. Next, the separation and detection of proteins was explored. As these peaks are somewhat wider, an integration time of 0.25 s/spectrum was judged to be sufficient. Figure 10 shows the TIC obtained from the separation of ubiquitin, myoglobin, and cytochrome c, each at a quantity of 28 fmol. At the higher field strength of -450 V/cm, the separation is still more than adequate to fully resolve the components, and the analysis time has been reduced to 125 s. Figure 11 shows a single, complete spectrum underneath each peak for the separation acquired with -450 V/cm, representing only 0.25 s of data. These spectra provide more than adequate information in order to deconvolute the molecular mass of each protein.

Figure 10. Effect of CE electric field strength on TICs from a protein separation (amine-coated column) using the TOF instrument.

Figure 9. Single scan for each peak in the TOF TIC data in Figure 8 (0.125 s/spectrum).

CONCLUSIONS The purpose of this work has been to demonstrate the advantages of using a TOF mass detector on-line with CE separations of peptides and proteins. When total separation times and peak widths can be measured in terms of a few seconds, the scan speed offered by this type of mass analyzer becomes mandatory. The observed results clearly indicate that the slower scanning speeds of other types of mass spectrometers, such as quadrupoles, are unsuitable in these particular applications. With experiments involving the use of a coated CE column, the peak widths and migration times were found to scale almost exactly inversely with the increasing applied electric field strength. Since the voltage applied is already at a practical maximum, even faster separations with narrower peak widths may be developed if the CE column length can be reduced along with the minimum distance from the CE column entrance to the mass spectrometer. This distance is currently the limiting factor on applied electric field strength. Additional encouragement toward this end is provided by experimental results that show that the relationship

Figure 11. Single scan for each peak in the TOF TIC data at -450 V/cm in Figure 10 (0.25 s/spectrum).

between the applied potential and the measured CE current for the buffer/sheath flow system used in these experiments is linear and thus not a cause of significant Joule heating. ACKNOWLEDGMENT This work was supported by the NIH under Grant 2 R44 RR09252-02. Received for review September 29, 1995. February 8, 1996.X

Accepted

AC9509824 X

Abstract published in Advance ACS Abstracts, March 15, 1996.

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