Anal. Chem. 1997, 69, 2908-2913
Protein Digest Analysis by Pressurized Capillary Electrochromatography Using an Ion Trap Storage/ Reflectron Time-of-Flight Mass Detector Jing-Tao Wu, Peiqing Huang, Michael X. Li, and David M. Lubman*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109
Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-offlight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-offlight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments. Capillary electrochromatography (CEC) is a novel liquid chromatographic separation method that uses electroosmotic flow (EOF) to transport solvent in a reversed-phase column. The use of EOF generates a nearly flat flow profile and a more uniform flow velocity distribution among all channels in a packed bed, which reduces the band broadening caused by transchannel diffusion and eddy diffusion. In addition, EOF allows the use of smaller packing materials, which further improves separation efficiency. Since the first report on CEC in 1974,1 the high separation efficiency of CEC has been demonstrated by several groups.2-4 Although it has a higher separation efficiency compared with that of conventional HPLC, CEC has been slow to develop due to various instrumental difficulties. In practice, it is difficult to obtain (1) Pretorius, V.; Hopkins, B. J.; Schieke, J. D. J. Chromatogr. 1974, 99, 2330. (2) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-16. (3) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317-28. (4) Lelie`vre, F.; Yan, C.; Zare, R. N.; Gareil, P. J. Chromatogr. 1996, 723, 14556.
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stable flow conditions since bubbles caused by Joule heating are easily formed during separation. Recently, the use of a supplementary pressure has been reported to stabilize the flow conditions in CEC, where bubble formation could be suppressed.5 The use of pressurized CEC (pCEC) does provide a very special advantage for the separation of charged species. The separation mechanism in CEC for neutral analytes is the solute partition between the stationary phase and the mobile phase, as in conventional HPLC. The mechanism for charged species, however, is more complicated, since both partition and electrophoresis will contribute to the separation. In CEC experiments without a supplementary pressure, the solvent flow results only from the EOF, which is proportional to the applied voltage. A change in the electrophoretic migration rate is always accompanied by a change in the mobile phase flow rate. In a pCEC system, however, the electrophoretic migration rate and the mobile phase flow rate can be optimized independently. This becomes possible since the applied voltage and the supplementary pressure are available as two tunable parameters that can be used to achieve enhanced separation performance that is otherwise difficult to obtain using either the HPLC or capillary electrophoresis mode in itself. To date, most of the CEC work has been focused on the separation of neutral compounds such as aromatic hydrocarbons, in which the analytes have no electrophoretic mobility. The separation of proteins and peptides by CEC has been reported by only a few groups, and in these, peptide mixtures consisting of one or two components have been successfully analyzed.6,7 The separation of relatively complex mixtures such as protein digests has not been reported. Indeed, the separation of a protein digest, especially when coupled with a mass detector, is the method of choice to study the sequence and posttranslational modifications of a protein. Because of its compatibility with the flow rate required by the electrospray ionization method, CEC can be directly interfaced to a mass spectrometer via electrospray ionization to provide the molecular weight of protein digest products and structural information via MS/MS. The use of mass spectrometers as on-line detectors for CEC has been reported recently by several groups.6-9 (5) Smith, N. W.; Evans, M. B. Chromatographia 1994, 38, 649-57. (6) Schmeer, K.; Behnke, B.; Bayer, E. Anal. Chem. 1995, 67, 3656-8. (7) Dekkers, S. E. G.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1995, 712, 201-9. (8) Gordon, D. B.; Lord, G. A.; Jones, D. S. Rapid Commun. Mass Spectrom. 1994, 8, 544-8. (9) Lane, S. J.; Boughtflower, R.; Paterson, C.; Morris, M. Rapid Commun. Mass Spectrom. 1996, 10, 733-6. S0003-2700(97)00183-2 CCC: $14.00
© 1997 American Chemical Society
The high efficiency and relatively fast speed achieved in CEC provide a challenge for a mass spectrometer when used as an on-line detector. A rapid, full-mass range sampling speed is required in order to accurately capture the high quality of fast on-line separations. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer developed in our laboratory has proved to be a rapid and sensitive on-line detector for fast separations in previous work.10-12 This device uses a quadrupole ion trap as a front end storage device, which converts a continuous electrospray beam into a pulsed beam for time-of-flight analysis. The storage property of the ion trap provides ion integration for low-intensity signals, while the nonscanning property of the timeof-flight mass spectrometer provides high sampling speed and high sensitivity. In previous work, the use of open-tubular column (OTC) CEC with an IT/reTOF mass detector for ultrafast separation has been reported,12 in which a peptide mixture and a protein digest were successfully separated within a short analysis time. The loadability for the OTC CEC system is in the low femtomole range due to the extremely small size of the column used. In this work, we report the use of a packed-column pCEC/IT/reTOF-MS system for protein digest analysis. At least a 1000-fold increase in sample loadability was achieved compared with that of the OTC CEC. It was shown both theoretically and experimentally that pressure and voltage can be used to optimize a separation, as demonstrated in the separation of a bovine cytochrome c digest. Also, a peptide mixture has been readily separated with relatively high content of the organic component. In addition, the separation of a chicken ovalbumin digest demonstrates the potential of this method as an efficient separation technique for relatively complex mixtures. EXPERIMENTAL SECTION Column Preparation. The pCEC columns were prepared from fused silica capillaries (180 µm i.d. × 360 µm o.d., Polymicro Technology, Phoenix, AZ) packed with 3 µm C-18 silica gel (courtesy of Vydac, Hesperia, CA). The use of a relatively large diameter of the CEC column allowed enhanced loadability and easy preparation of end-column frits. A slurry of methanol and the packing material was prepared at a ratio of 1:10 (v/w) and packed to a pressure of 5000 psi for a 12 cm long column. Each column end was assembled into a Valco microbore column endfitting, with a very small amount of glass wool used as the frit. The frits prepared in this method were found to be much more durable and reproducible compared to frits prepared with the silica gel-sintered method. The thickness of the frits was about 0.1 mm, resulting in a small dead volume. The column end-fittings served to provide a point of electrical contact. pCEC Apparatus. The inlet end of the pCEC column was connected to a Valco six-port injection valve, where the high separation voltage was applied, while the outlet end of the column was grounded and connected to an electrospray source via a 50 µm i.d. fused silica capillary. A Varian Star 9012 solvent delivery pump (Varian Associates, Inc., Walnut Creek, CA) was used to provide the supplementary pressure where the solvent gradient and solvent split were performed before the injection valve. A grounding device was installed on the transfer capillary between (10) Qian, M. G.; Lubman, D. M. Anal. Chem. 1995, 67, 2870-7. (11) Wu, J.-T.; Qian, M. G.; Li, M. X.; Liu, L.; Lubman, D. M. Anal. Chem. 1996, 68, 3388-96. (12) Wu, J.-T.; Huang, P.; Li, M. X.; Qian, M. G.; Lubman, D. M. Anal. Chem. 1997, 69, 320-6.
the split valve and the injection valve to protect the HPLC pump. For gradient elution, 0.07% trifluoroacetic acid (TFA) in water was used as solvent A, and 0.07% TFA in acetonitrile was used as solvent B. The purpose of using a relatively low concentration of TFA was to reduce the Joule heating effect. No effect on the elution of the peptides has been observed in HPLC experiments by using these solvents with a relatively low TFA concentration. Sample stacking was performed by running solvent A for 2 min before each separation. This step allowed the sample to be focused at the beginning of the stationary phase.13 The purpose of this sample stacking was to reduce the band broadening caused by the injection volume and lower the concentration limit of detection. For most of the experiments, a sample concentration of 5 × 10-6-1 × 10-5 M was used, with an injection volume of about 1-1.5 µL, corresponding to a sample load in the low picomole range. This sensitivity was comparable to that of the capillary LC/MS work reported from our laboratory,10 but it was not as high as that obtained from our OTC CEC/MS work, where low femtomole level detection has been achieved due to the small dimension of the column.12 Mass Spectrometer. An IT/reTOF MS as described in previous work10 was used in these experiments as an on-line detector for CEC separations. This device consists of a quadrupole ion trap (Model C-1251, R. M. Jordan Co., Grass Valley, CA) interfaced to a reflectron time-of-flight mass analyzer (Model D1450). Ions generated from the electrospray source were introduced into an atmospheric pressure interface through a heated stainless steel capillary (0.5 mm i.d., 140 °C). The ions traversing the interface were focused by a coaxial cylindrical lens (+100 V) and subsequently passed through a skimmer orifice (325 µm) into the high-vacuum chamber. The ions were then focused through an Einzel lens into the ion trap. The ions were stored in the ion trap under a preset rf voltage of 1250 V on the ring electrode for a period of 250 ms, corresponding to a detection speed of 4 Hz in these experiments. The ions were then ejected by a dc pulse on the endcap of the ion trap into the time-of-flight device for analysis. The ions were mass separated by the reflectron TOF device and detected by a 25 mm triple-microchannel plate detector (Model C-2501, R. M. Jordan Co.). The data system used in these experiments was based on a 250 MHz highspeed transient recorder (Model 9846, Precision Instruments Inc., Knoxville, TN) embedded in a Pentium 66 MHz PC compatible computer (Model P5-66, Gateway 2000, North Sioux City, SD).14 This system is capable of conducting data acquisition and storage at rates of over 10 Hz for a minimum of 30 min, for individual mass spectra containing 16 000 data points with 10 ns resolution. A sampling time window width of 150 µs, which corresponds to a mass range from 200 to ∼1500 Da, was used for all studies in this work. Materials. All peptide and protein samples, acetonitrile, and TFA were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. Trypsin was purchased from Promega (Madison, WI). Water used to prepare CEC solvent was generated with a Milli-Q water purification system (Millipore Corp., Bedford, MA). Chicken ovalbumin was first denatured, reduced, and alkylated in a standard manner before (13) Liao, J.-L.; Chen, N.; Ericson, C.; Hjerten, S. Anal. Chem. 1996, 68, 346872. (14) Qian, M. G.; Wu, J.-T.; Parus, S.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1996, 10, 1209-14.
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Figure 1. TICs of the separation of a six-peptide mixture using 25% acetonitrile isocratic elution with sample injections of 10 pmol. Column length, 12 cm. According to the elution order, the six peptides are bradykinin, angiotensin II, angiotensin I, Met-enkephalin-Arg-Phe, neurotensin, and substance P. Conditions: (a) 100 bar supplementary pressure, no applied voltage; (b) 100 bar supplementary pressure, 3000 V applied voltage.
digestion.15 For tryptic cleavage, 100 µg of protein was incubated for 24 h at 37 °C with a protein-to-enzyme ratio of 50:1 (w/w) in 50 mM NH4HCO3 solution at pH 8.2. The digested materials were then vacuum-dried to remove the salt and reconstituted in solvent A to a concentration of 1 × 10-5 M original protein. RESULTS AND DISCUSSION Pressurized CEC is a technique that provides distinct advantages for the separation of peptide mixtures. The superposition of a plug flow profile with a pressure-driven parabolic flow reduces band broadening and allows the use of small packing materials, resulting in high separation efficiency. The use of a supplementary pressure makes it possible to achieve enhanced separation performance using the electrical field. An important characteristic of the capacity factors of peptides is that small changes in isocratic concentrations of the organic component in the mobile phase result in dramatic changes in (15) Stone, K. L.; LoPresti, M. B.; Crawford, J. M.; DeAngelis, R.; Williams, K. R. In A Practical Guide to Protein and Peptide Purification for Microsequencing; Matsudaira, P. T., Ed.; Academic Press: San Diego, CA, 1989; p 33.
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peptide retention times. This is related to the fact that peptides are generally hydrophobic but also carry net positive or negative charges, so the interaction between peptides and reversed-phase packings is based mainly on an adsorption/desorption mechanism and on partition only to a limited extent.16 For the separation of a peptide mixture using the isocratic mode, the range of the organic component concentration is extremely narrow. Therefore, a gradient elution is necessary for the separation of a peptide mixture using conventional HPLC. In pCEC, the electrophoretic migrations of the peptides provide additional dispersions, which are virtually independent of the organic component concentration in the mobile phase; therefore, it is relatively easier to separate a simple peptide mixture using the isocratic mode, as demonstrated in Figure 1. In Figure 1 are shown the total ion chromatograms (TICs) of a separation of a six-peptide mixture with pCEC using the isocratic elution mode with 25% acetonitrile in the mobile phase before (Figure 1a) and after (Figure 1b) applying a voltage. A 12 cm long column was used, with a supplementary pressure of 100 bar. In Figure 1b, the sample elution time was about 10 min shorter than that in Figure 1a, even though these experiments were performed under the same pressure. This is the result of a combination of EOF and electrophoretic migration. Before any voltage was applied, the peaks were broad and partially resolved, since the capacity factors of some of the peptides were close to each other at this acetonitrile concentration. After a 3000 V voltage was applied across the column, all six peptides were resolved as a result of the higher separation efficiency and the additional dispersion arising from the difference in electrophoretic mobilities of the peptides. Fast separation of peptide mixtures by pCEC in the isocratic mode can be achieved, even at high organic solvent content (40% acetonitrile). For the separation of charged species, such as peptides, the use of a supplementary pressure in CEC allows the tuning of the elution of the components. This is difficult to achieve in CEC without a supplementary pressure. The capacity factor k′of a peptide in CEC is, by definition,
k′ )
tR - to νo - νR ) to νR
(1)
where tR and to are the retention times for the peptide and an unretained solute, respectively, and νR and νo are the migration rates of the peptide and an unretained solute, respectively. In CEC without a supplementary pressure, the solvent flow in the column comes only from the EOF, whose flow rate νEOF is proportional to the applied electrical field E, using the same column and solvent. Therefore,
νo ) νEOF ) aE
(2)
where a is a constant for the same column and solvent. The migration rate of a peptide in CEC is the sum of the migration rates arising from the EOF and the electrophoretic (16) Mant, C. T.; Lorne Burke, T. W.; Hodges, R. S. Chromatographia 1987, 24, 565-72.
migration4 and thus is given by
νR )
νEOF + νele 1 + k′LC
(3)
where k′LC is the capacity factor of the peptide in the same column operated in the conventional HPLC mode (with a back-pressure only), and νele is the electrophoretic migration rate of the peptide, which is proportional to the applied electrical field using the same column and solvent, and is therefore given by
νele ) bE
(4)
where b is a constant for the same peptide using the same column and solvent. Combining eqs 2-4 gives
νR )
a+b E ) cE 1 + k′LC
(5)
where c ) (a + b)/(1 + k′LC). c is a constant for the same peptide with the same column and solvent. Therefore, the electrochromatographic capacity factor of a peptide in CEC without a supplementary pressure can be written as
k′)
aE - cE a - c ) cE c
(6)
Surprisingly, the electrical field E term disappears from the above final expression of the capacity factor. Therefore, in theory, for CEC separations without a supplementary pressure, it is impossible to tune a capacity factor of a peptide by the applied electrical field. Likewise, it is impossible to optimize the elution of peptides using the electrical field, since it is just a ratio of capacity factors. In practice, slight changes in the capacity factors of peptides at different electrical fields could possibly occur due to the electrically induced concentration polarization at the stationary surface17 and the column temperature changes that might occur due to Joule heating. In pCEC separations of peptides, because of the existence of a pressure-driven flow, eq 2 becomes
νo ) νEOF + νpre ) aE + λP
(2′)
where νpre is the pressure-driven flow rate, P is the supplementary pressure, and λ is a constant for the same column and solvent. Accordingly, eqs 5 and 6 become
νR ) cE + k′)
λ P 1 + k′LC
(a - c)E + dk′LCP cE + dP
(5′)
(6′)
where d ) λ/(1 + k′LC), which is a constant for the peptide using the same column and solvent. Thus, in a pCEC system, the capacity factor is no longer independent of the applied electrical field. Also, an additional variable, the supplementary pressure, (17) Basak, S. K.; Velayudhan, A.; Kohlmann, K.; Ladisch, M. R. J. Chromatogr. 1995, 707, 69-76.
Figure 2. TICs of the separation of a bovine cytochrome c digest using a 20 min, 0-50% acetonitrile gradient with sample injections of 8 pmol corresponding to the original protein. Column length, 6 cm. Column operation conditions: (a) HPLC mode with a back-pressure of 90 bar; (b) 1000 V applied voltage with 50 bar supplementary pressure; (c) 1400 V applied voltage with 50 bar supplementary pressure; and (d) 600 V applied voltage with 70 bar supplementary pressure.
is now available. Indeed, the tuning of the elution can be achieved by properly adjusting the applied field and the supplementary pressure, as demonstrated in Figure 2. In Figure 2 are shown the TICs of the separation of a bovine cytochrome c digest. A 6 cm long column with a gradient elution was used in these experiments. In Figure 2a, no separation voltage was applied, and the separation was performed in the normal HPLC mode, with a back-pressure of 90 bar. The use of a short column in the HPLC mode made it difficult to resolve all the components in the digest, as indicated by the peak marked by an arrow, which contains two coeluting components. In Figure 2b, a 1000 V voltage was applied on the column, and the backpressure was reduced to 50 bar. The purpose of reducing the back-pressure after applying a voltage is to maintain a relatively constant mobile phase flow rate, since this flow rate could dramatically affect a separation process. Even with a reduced back-pressure, all the components eluted faster in Figure 2b than in Figure 2a, due to the contribution from EOF and electrophoretic migration. The peaks are sharper in Figure 2b, indicating an increase in separation efficiency. The two coeluting components in the peak marked by an arrow in Figure 2a were separated to base line in Figure 2b (marked by 3 and 4), mainly due to the Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
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Figure 3. TIC of the separation of a tryptic digest of chicken ovalbumin with a sample injection amount of 12 pmol corresponding to the original protein. Column length, 6 cm. Conditions: 20 min, 0-40% acetonitrile gradient; 1000 V applied voltage with 40 bar supplementary pressure. Table 1. Comparison of Calculated and Measured Tryptic Peptides of Chicken Ovalbumin from pCEC/MS Analysis no.
tryptic peptides
calcd massa
determined massa,b
sequence
1 4, 5 5 6, 7 7 10 11 12 13 16 16, 17 17 18 20 21 23 24, 25 26 26, 27 27, 28 28 30 31 32 33 33, 34 34
1-16 47-55 51-55 56-61 59-61 105-110 111-122 123-126 127-142 182-186 182-189 187-189 190-199 219-226 227-228 264-276 277-279 280-284 280-286 285-290 287-290 323-339 340-359 360-369 370-381 370-385 382-385
1709.0 1080.2 602.7 781.0 408.5 779.8 1465.8 579.7 1687.8 631.7 996.1 364.4 1209.3 821.9 277.4 1581.7 405.5 646.8 924.2 813.0 535.6 1773.9 2009.1 1190.4 1345.6 1750.1 404.5
1709.6 1079.7 602.9 781.4 408.4 780.1 1466.3 579.7 1687.5 631.6 995.9 364.5 1209.0 821.7 277.6 1581.3 405.4 646.8 924.4 813.1 535.5 1774.2 2008.5 1190.2 1345.3 1749.5 404.4
GSIGAASMEFCFDVFK DSTRTQINK TQINK VVRFDK FDK IYAEER YPILPEYLQCVK ELYR GGLEPINFQTAADQAR GLWEK GLWEKAFK AFK DEDTQAMPFR VASMASEK MK LTEWTSSNVMEER KIK VYLPR VYLPRMK MKMEEK MEEK ISQAVHAAHAEINEAGR EVVGSAEAGVDAASVSEEFR ADHPFLFCIK HIATNAVLFFGR HIATNAVLFFGRCVSP CVSP
a
Average masses. b Average of all charge states observed.
electrophoretic separation of these two components. However, the first two peaks in Figure 2a (marked by 1 and 2) migrated into one peak in Figure 2b (marked by an asterisk), since, apparently for these two particular fragments, the electrophoretic 2912 Analytical Chemistry, Vol. 69, No. 15, August 1, 1997
Figure 4. Mass spectra corresponding to the the peak marked by an arrow in Figure 3. (a) Fragment TQINK, m/z ) 603.9, eluted at 12.95 min. (b) Fragment VYLPR, m/z ) 647.8, eluted at 13.01 min.
separation is in the opposite direction of the separation resulting from partition. A further increase in the applied voltage resulted in even faster separations (1400 V, Figure 2c), but the peak marked by the asterisk is still unresolved. With an adjustable supplementary pressure pCEC, the applied voltage can be tuned over a wide range, as shown in Figure 2d, where the separation was performed at a relatively low voltage of 600 V, with a supplementary pressure of 70 bar. At this specific pressure and voltage, all the four peaks were clearly resolved. It should also be noted that, without this supplementary pressure, the EOF generated by this relatively low voltage would be too slow to perform a chromatographic separation. Even though the above TICs were obtained with the same sample load, the S/N ratio decreased as the applied voltage increased. This was probably due to the instability of the electrospray signal caused by small bubbles. The use of a supplementary pressure greatly suppressed bubble formation during the separation; however, reduced bubble formation does occur near the end of the column and in the transfer capillary after the column, where the pressure approaches 1 atm. Typical base line peak widths in the above TICs are around 6-8 s. A 4 Hz full-mass sampling rate was used, which corresponded to about 30 recorded mass spectra for a typical peak. The enhanced separation performance achieved in pCEC allowed the use of short columns, which resulted in reduced analysis time. Also, the electrophoretic migrations of peptides, which were in the same direction as the mobile phase flow in these experiments, enhanced the separation speed. Therefore, relatively fast separations have been achieved. However, this separation speed was not significantly better than that obtained in the work reported using conventional HPLC.18 Further work
needs to be done to optimize the separation conditions to enhance the separation speed of pCEC in the future. The IT/reTOF mass spectrometer used in these experiments successfully provided rapid and sensitive detection for these pCEC separations, which not only maintained the high separation efficiency but also provided correct molecular weight information. Indeed, an advantage of using a mass spectrometer as an on-line detector is that the molecular weight information obtained can be used to identify coeluting components during the separation. When this capability is combined with the enhanced separation performance of pCEC, the pCEC/MS system can be used to analyze complex protein digests in a relatively short time, as demonstrated in Figure 3, where the TIC of a tryptic digest of chicken ovalbumin is shown. Over 20 peaks were resolved in the TIC in about 15 min. With the assistance of the mass detector, 27 fragments could be clearly identified. The calculated and the determined masses of these fragments are listed in Table 1. In Figure 4 is shown one example of using the mass detector to identify coeluting components in the analysis of this complex digest. The mass spectra shown in Figure 4 correspond to two coeluting components in the peak marked by an arrow in Figure 3. Even though the extracted ion profiles for these two components showed that the difference in the retention time was only about 3 s, mass spectra which contained a single component could be achieved due to the fast sampling rate of the mass detector. (18) Stoney, K.; Mock, K.; Sanders, M. Proceedings 41st ASMS Conference Mass Spectrom Allied Topics, San Francisco, CA, May 31-June 4, 1993; p 582a.
These mass spectra were single spectra without any averaging using a trap storage time of only 0.25 s, yet they both exhibited good S/N for mass identification. Those two components are identified as fragments TQINK and VYLPR, respectively. CONCLUSIONS Pressurized CEC is a very useful separation technique for the analysis of protein digests. It has been shown both theoretically and experimentally that tuning of the elution of peptides can be achieved only in CEC with a supplementary pressure. The combination of the enhanced separation performance from pCEC and the molecular weight information from the IT/reTOF mass spectrometer makes the pCEC/MS system capable of analyzing relatively complex protein digests in a short time. ACKNOWLEDGMENT We gratefully acknowledge support of this work by the National Institutes of Health under Grant No. 1R01GM49500 and the National Science Foundation under Grants No. BIR-9223677 and BIR-9513878. We also thank Dr. Mark G. Qian of Chiron Corp. for helpful discussions. Received for review February 13, 1997. Accepted May 22, 1997.X AC970183G X
Abstract published in Advance ACS Abstracts, July 1, 1997.
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