Anal. Chem. 1995, 67,2870-2877
Analysis of Tryptic Digests Using Microbore HPLC with an Ion Trap Storage/Reflectron Time-of-Flight Detector Mark 0. Qianand David M. Lubman* Department of Chemistry, The University of Michigan, Ann Atbor, Michigan 48109
An electrosprayionization source interfacedto an ion trap storage/rdectron time-of-flight mass spectrometer is evaluated as a rapid, sensitive detector for microbore HPLC. Using the total ion storage capabilities of the trap over a broad mass range, total ion chromatograms of tryptic digests of bovine cytochromec and bovine /3-casein are obtainedfollowing microbore HPLC separationswith samples in the low picomole range. The digests are analyzed with the aid of software developed in our laboratory, which can display the selected ion chromatogram (SIC) for each chromatographic peak. The SIC mode can be used for enhancementof the S/N and for identification of a chromatographic peak with a particular mass. In addition, the mass spectrum corresponding to each chromatographic peak can be displayed to check for unresolved chromatographiccomponents. The use of mass spectrometry as a detector for on-line separations of mixtures in solution has recently become an important method for characterizationof proteins and peptides.'-1° In particular, mass spectrometric detection allows for differentiation of mixtures eluting from separation methods based not only on their separation time but also on their mass and fragmentation patterns. Its use as a detector for on-line separations has become especially significant with the development of electrospray ionization as a means of producing molecular ions of large proteins and peptides from s ~ l u t i o n . ~ Indeed, ~ - ~ ~ ESI has been used for (1)Huang. E. C.; Henion, J. D. J. Am. SOC.Mass Spectrom. 1989,1 , 158-165. (2) Covey, T. R.;Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63,11931200. (3) Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63,732-739. (4)Suter, M.; Dague, B. B.; Moore, W. T.; Lin, S. N.; Caprioli, R. M. J. Chromatogr. 1991,553, 101-116. (5)Heath, T.G.: Giordani, A B. J. Chromatogr. 1993,638,9-19. (6) McLuckey, S. A;VanBerkel, G. J.; Glish, G. L.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991,63,375-383. (7)Hopfgartner, G.; Bean, K; Henion, J.; Henry, R.J.Chromatogr. 1993,647, 51-61. (8) Emmett, M. R;Caprioli, R. M. J. Am. SOC.Mass Spectrom, 1994,5, 605613. (9)Fang, L.;Zhang, R;Williams, E. R.; Zare, R N. Anal. Chem. 1994, 66, 3696-3701. (10)Kassel. D. B.; Shushan, B.; Sakuma, T.;Salzmann, J. P.Anal. Chem. 1994, 66.236-243. (11) Fenn, J. B.; Mann, M.; Meng, C. K; Wong, S. F.; Whitehouse, C. M. Science 1989,246,64-71. (12)Fenn, J. B.; Mann, M.; Meng, C . K; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990,9, 37-70. (13) Smith, R D.; Olivares, J. A; Nguyen, N. T.; Udseth, H. R Anal. Chem. 1988, 60,436-441. (14)Barinaga. C. J.; Edmonds, C. G.; Udseth, H. R.;Smith, R D.Rapid Commun. Mass Spectrom. 1989,3,160-164.
2870 Analytical Chemistry, Vol. 67, No. 77, September 7, 7995
coupling on-line separations with mass spectrometry for highperformance liquid chromatography (HPLC) ,2q7 capillary electrophoresis (CE) ,4,9 and m i c r o b ~ r e and ~ * ~capillary ~ ~ ~ ~HPLC.3-5,10 ~~ Although ESI has been interfaced to various types of mass spectrometers, a key feature of ESI is the ability to produce multiply-charged ions of large proteins. The result is that large ions can be detected at a relatively modest madcharge, and so inexpensive quadrupole mass spectrometers have become widely used detectors for chromatographic separations. An inherent problem in interfacing on-line separations with scanning mass spectrometers such as quadrupole and sector devices is the duty cycle of the experiment. In capillary electrophoresis, for example, the duration of the peaks may be on the order of several sec0nds,'~J6while in the case of capillary and microbore HPLC separations, peak widths may be on the order of 20 s or less. The use of scanning mass spectrometers inherently limits the sensitivity since it detects only one mass at a time, while the ion signal generated during peak elution is continuous. Most of the ion signal is thus lost due to the poor duty cycle of these scanning instruments. Moreover, in the case of very narrow chromatographic peaks, a scanning mass spectrometer may provide a skewed response since the concentration changes during the scan time. In recent work, the ion trap mass spectrometer (TITvIS)~J~and Fourier transform mass spectrometer (FT-ICR),18-20 which can collect the total ion current over a broad mass range, have been interfaced to an ESI source. The ion trap has appeared to be particularly promising on the basis of the excellent sensitivity achieved through the high duty cycle and its ion storage and integration capabilities. In addition, the trap operates at an elevated pressure (10-2-10-4 Torr), which is conveniently interfaced to atmospheric pressure interfaces such as those used in electrospray. Also, the use of high pressure in the trap results in collisional dissociation of unwanted solventanalyte clusters.6 The trap method should also be able to accurately detect the eluents of even the fastest separations. However, there still remain difficulties in terms of scanning high mass out of the trap without using auxiliary radio frequency (rf) voltage, which may result in a deterioration of the mass accuracy, (15)Jorgenson, J. W.; Lucas, K D. Science 1983,222,266-272. (16)Burlingame, A L.;Boyd, R. K; Gaskell, S. J. Anal. Chem. 1994,66,634R683R (17)Mordehai, A V.; Hopfgartner, G.; Huggins, T. G.; Henion, J. D. Rapid Commun. Mass Spectrom. 1992,6,508-516. (18)Hofstadler, S.A;Laude. D. A. J. Am. SOC. Mass Spectrom. 1992,3,615623. (19)Loo, J. A,; Quinn. J. P.; Ryu,S. I.; Henry, K D.; Senko, M. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. US.A. 1992,89, 286-289. (20)Loo, R;Loo, J. A.;Udseth, H. R;Fulton, J. L.;Smith. R.D. Rapid Commun. Mass Spectrom. 1992,6,159-165. 0003-2700/95/0367-2870$9.00/0 0 1995 American Chemical Society
resolution, and scan speed. Alternatively, FT-ICR can provide total out rate. The duty cycle here is >99% and is due to the deadion collection of electrospray-produced ions over a broad mass time of the dc pulse-out and TOF flight time as compared to scanning ion traps, where hundreds of milliseconds may be range with very high resolution. However, this high resolution is achieved only following a sufficiently long ion storage time, needed to scan a spectrum and the resulting duty cycle may be which will ultimately liiit its capability as a fast on-line detector. significantly lower. This is especially the case for the slow scan mode of ion traps required to obtain enhanced resolution in these In addition, the high cost of FT-ICR and the pumping capabilities devices. In addition, the dc ejection mode of the IT/reTOFMS required for atmospheric pressure interfaces needed to maintain obviates the need for sophisticated software to accurately scan the low pressure required by the FT-ICR will limit its applications the mass range of traps in the mass-selective instability mode. as a widely used detector. An additional advantage of the IT/reTOFMS as compared to An alternative means of achieving fast on-line detection with the TOFMS is that the storage properties of the trap provide ion separation methodology is the time-of-flight mass spectrometer integration of low-intensity signals, thus enhancing the sensitivity (TOFMS). The TOFMS has the key advantages of high speed, for detection. It may also provide the capability for selective wide mass range, simplicity, and high sensitivity. The TOFMS ejection of unwanted background ions and storage of target ions is a nonscanning device which can measure a complete mass of interest based upon resonance ejection methods and ultimately spectrum over an extended mass range following every injection to MS/MS capabilitiesbased upon these m e t h o d ~ . ~ sThe ~ ~ trap -~~ pulse of ions. Thus, this device can provide rapid detection and can also operate at higher pressure than TOF devices, allowing high duty cycle for separation methodology, resulting in accurate for easy interfacing to atmospheric pressure sources and collipeak shapes and high sensitivity. In addition, relatively high sional breakup of cluster ions formed with the solvent. In addition, resolution and mass accuracy can be provided by a reflectron the storage properties of the IT may be combined with the highTOFMS in a rather simple and inexpensive instrument. Further, resolution properties of the reTOFMS to produce an instrument the high mass range of a TOF device makes it capable of capable of a resolution of several thousand, as shown in previous monitoring a broad range of charge states in an ESI-based mass ~ork.2~~~~ spectrometer. In the work presented herein, we demonstrate the capabilities There have been several recent efforts to interface ESI and of the ESI/IT/reTOFMS as a fast and sensitive detector for other continuous ion beam sources to TOFMS. The main problem microbore chromatography. Microbore HPLC separations of here is that the TOFMS requires an ion pulse or start time to tryptic digests of various proteins are separated and interfaced to achieve time resolution, so an appropriate method is required to an ion spray version of the ESI source, operating at between 40 convert a continuous ion beam into pulsed ion packets. One such and 50 pL/min. Ion storage times of 500 ms are used to achieve method uses a pulsed orthogonal extraction geometry, which has long-term ion integration for enhanced sensitivity of lowachieved high resolution and rapid d e t e c t i ~ n ? * However, ~ ~ - ~ ~ in concentration samples, with a resulting high duty cycle in order to achieve a high duty cycle with this device, a high pulsed detection. Using the total ion storage capabilitiesof the trap, total extraction repetition rate (>2000 Hz) must be used. This high ion chromatograms of tryptic digests of cytochrome c and bovine repetition rate, in combination with the potentially large record ,!?casein are obtained with detection limits in the low picomole length involved and the speed with which the acquired data must range. In addition, using software developed in our laboratory, be stored, requires specially designed circuitry and software or the selected ion chromatogram can be displayed for both the use of ion counting. In addition, the dead-time restraint of enhancement of the S/N and idenscation of a chromatographic commonly used MCP detectors may also limit the operation of peak with a particular mass. Also, the mass spectrum of each the TOFMS in this high-repetition-rate orthogonal extraction chromatographic peak can be displayed in order to search for mode. Also, it should be noted that a major limitation of this unresolved chromatographic peaks. The entire data file can configuration is that there is no capability available for MS/MS ultimately be displayed as a 3D spectrum in analogy to a 2D gel or selective ejection of unwanted ions as in ion trap devices. used for separations in the biological sciences. The ability to In recent work, we have demonstrated the use of a combination detect even small differences in protein structures using this ion trap storage/reflectron time-of-flight mass spectrometer is discussed. (IT/reTOFMS) as a means of interfacing ESI to a TOF d e v i ~ e . ~ ~methodology ~~~ The IT/reTOFMS uses an ion trap as a front-end storage device prior to mass separation and identification by the reTOFMS. In EXPERIMENTAL SECTION this device, the ions are stored by an rf-only voltage on the ring The experimental setup consists of an HPLC separation system electrode and after a delay are simultaneously ejected by a dc interfaced to an electrospray ionization source with detection using pulse into the reTOFMS for analysis. The dc ejection pulse the IT/reTOF system. A gradient elution liquid chromatography provides the start time for the reTOFMS. The trap thus serves system using reversed phase columns was initially used to as a means of converting a continuous electrospray beam into a optimize separation of mixtures of peptides and protein digests. pulsed beam for analysis by TOF mass spectrometry. An The system was setup to accommodate either microbore or important advantage of this method is that the storage properties narrowbore column HPLC separations. The liquid eluent of the of the trap allow a high duty cycle to be achieved with a low pulseseparation is then delivered through a fused silica capillary tube into an electrospray assembly, where the sample is ionized. The (21) Boyle, J. G.; Whitehouse, C. M. Anal. Chem. 1992,64, 2084-2089. (22) Mirgorodskaya, 0. A; Shevchenko, A A; Chernushevich, I. V.; Dodonov, electrospray source utilizes gas on-axis nebulization methods or A F.; Miroshnikov, A. I. Anal. Chem. 1994,66,99-107. ion spray to accommodate higher flow rates. The droplets (23) Verentchikov, A N.; Ens, W.; Standing, K G. Anal. Chem. 1994,66, 126133. (24) Michael, S. M.; Chien, B. M.; Lubman, D. M. Anal. Chem. 1 9 9 3 , 6 5 2 6 1 4 2620. (25) Chien, B. M.; Lubman, D. M. Anal. Chem. 1994, 66, 1630-1636.
(26) Cooks, R G.; Kaiser, R E. J. Acc. Chem. Res. 1990,23, 213-219. (27) March, R; Hughes, R Quadrupole Storage MQSSSpectromety; Wiley: New York, 1989.
Analytical Chemistty, Vol. 67, No. 17, September 1, 1995
2871
produced are then sampled through a heated stainless steel (ss) capillary to desolvate the droplets, leaving highly charged peptide ions. The ions are then injected into a differentially pumped interface, where the on-axis component of the ion beam passes through a skimmer into the mass spectrometer region. The ions are transported via a set of Einzel lens into the quadrupole ion trap storage device. The ions are stored and accumulated to an optimal sensitivity for detection of electrospray-produced ions. Ultimately, an extraction pulse is applied to the exit endcap, and the ions are ejected into the reTOFMS for detection and mass ' analysis. The use of the reflectron device provides the enhanced resolution observed in these experiments. The ions are detected by a triple 4@mmmicrochannel plate assembly, and a data system is used to digitize and process the resulting mass-detected chromatogram. Liquid Chromatography. A Star 9012 solvent delivery system (Varian Associates, Inc., Walnut Creek, CA) was operated at a iked flow rate of 200 pL/min for both microbore and narrowbore HPLC separations. In the case of microbore separations, a prime/purge valve located immediately before the injection valve was used to split the mobile phase flow with a ratio of 3:l. The synthetic peptide mixtures and tryptic digest samples were injected through a Valco Model C6W sample injector (Valco Instruments Co. Inc., Houston, nr),equipped with a 1@pL external sample loop. Microbore separations were accomplished with a l.@mm x 15" CIScolumn (5 pm, 300 A) from Alltech Associates, Inc. Oeertield, IL) at a flow rate of 50 pL/min. Effluents exiting the columns were monitored by a Star 9050 (Varian) variable wavelength UV detector at 214 nm, which was digitized through a l&bit ADC board embedded in a 486 PC compatible computer. The only mod~cationto the HPLC system was replacement of the original 4.5pL flow cell in the UV detector with a 60-nL microcapillary flow cell (Varian) in order to minimize the dead-volume during the UV monitoring process. All separations were camed out with 0.1%TFA in water as solvent A and 0.09% TFA in 9O:lO acetonitrile/water as solvent B. A linear gradient was employed for the LC/MS separations beginning with 100%of A, followed by ramping the B content to 80%in a period of 60 min unless otherwise stated. Under these conditions, most separation times were
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Figure 3. Selected ion chromatograms of cytochrome c tryptic peptides from 100-pmol injection with center masses at (a) mk634.4 for peak 1, (b) m/z 907.5 for peak 2, and (c) m/z 584.1 for peak 3.
There are some differences in the relative peak heights of the corresponding peaks in Figures 1 and 2, which is due mainly to the relative differences in UV absorption efficiency versus the detection of ion peaks by mass analysis. In addition, there is a rising baseline as a function of time for the chromatogram observed in the W detection. This is due to the absorption of the UV radiation by the increasing amount of acetonitrile in the acetonitrile/water gradient as a function of time. The increasing amount of acetonitrile does not affect the chromatogram obtained with the ESI/IT/reTOF detection significantly. Nevertheless, the chromatogram obtained via the TIC mode in the IT/reTOFMS is remarkably similar to that obtained using the W absorption detector. The high duty cycle provided by the total ion collection over a broad mass range by the IT/reTOFMS results in excellent detection limits for on-line analysis. Also, the nonscanning operation of the IT/reTOF allows rapid data collection and averaging for chromatographic separations, even for the fastest separations. In Figure 2b, the TIC chromatographic separation is detected with excellent S/N even at a level of 20 pmol of total injected sample of cytochrome c. Even at a level of Cpmol injection, there is still a discernible chromatogram obtained. However, the TIC detection limit for detection without loss of chromatographic information in this case is between 10 and 20 pmol, whereas for UV detection it is typically 1-5 pmol. It should be noted that the sensitivity observed is considerably higher than that for cytochrome c injected by continuous flow from a water/ methanol solution into the electrospray ionization source. The 2874 Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
main limitation to the detection limit in on-line analyses is the use of TFA and other organic buffers required to optimize the chromatographic separation. The recent development of alternate buffers may allow a further significant reduction in the detection limit. In order to enhance the S/N in the ion chromatograms, the ion signal is integrated over a long storage time and is signal averaged by the Precision Instruments digital acquisition board. In these spectra, the ions are being stored for 500 ms/cycle before being ejected into the reTOFMS for analysis. A total of four spectra are averaged before each point is plotted, where a point is plotted, every 2 s. The result is that at least 15 data points are involved in defining a chromatographic peak. The present limit to this data processing rate is the long trap storage time used to integrate the limited ion signal obtained at low concentrations. The use of a 50Gms storage time allows faster integration of the ion signal than that of random background ions. The use of long storage times provides a marked increase in S/N, which can be further signal-averaged over several pulses. The electrospray-produced ions are detected in these on-line experiments in the total ion storage mode, where ions of all masses over a broad mass range are stored by the trap and detected by the TOFMS. The chromatograms shown in Figure 2 are thus obtained for the total ion current. However, the data can be reprocessed by our computer software to provide the ion chromatogram for selected masses only. This procedure provides a selected ion chromatogram (SIC) for each mass in the spectrum detected. This capability is demonstrated in Figure 3 for the SIC of peaks 1, 2, and 3 of Figure 2a. This method allows us to correlate each chromatographic peak with one or more particular masses. Some of the identified fractions of cytochrome c tryptic digest with both measured and calculated masses are listed in
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mh Figure 5. Ma ss spectra of the cytochrome c tryptic fragments corresponding ct 3 the SICS shown in Figure 3. Table 1. Cor nparison of Calculated and Measured Tryptic Fragi nents of Bovine cytochrome c from the LCMS Analy si8
no.
fragm ent
12
1-' 7 9- 13 9--.22 14- 22 28- 38 39- 53 56- 72 74- 79 80- 86 80- 87 89- 99 92- 99 101- 104
4 4,5 5 8 9,lO 12 14 15 15,16 18,19 19 21
calcd mass
732.39 634.39 1633.82 1018.44 1168.62 1584.77 2009.95 678.38 779.95 907.54 1306.40 964.54 434.19
measd mass"
sequence
732.4 GDVEKGK 634.4 IFVQK 1634.1 IFVQKCAQCHTVEK 1017.9 CAQCHTVEK 1167.9 TGPNLHGLFGR 1583.9 KTGQAPGFSYTDANK 2010.1 GITWGEETLMEYLENPK 678.2 YIPGTK 779.5 MIFAGIK 907.5 MIFAGIKK 1306.7 G E E DLIAYLK 964.7 E D U Y L K 434.3 ATNE
Average IT inss c all charge states c the fragment observed. Table 1. In a ddition, since the background ions at other masses have been elii minated from the chromatogram, the S/N is greatly improved. TI his is demonstrated in Figure 3, where the S/N is very much ei ihanced, even for a minor peak (peak 2) in the ion chromatogram of Figure 2a. A lower limit of detection for cytochrome :of -1 pmol or less is routinely obtained using this methodology. , The improvement in sensitivity that can be achieved using the SI(2 mode is further demonstrated in Figure 4. This figure shows the SIC spectra for chromatographicpeaks 4,5, and 6 in the 4p:mol TIC spectrum of cytochrome c of Figure 2c. Although thc 2 S/N is relatively poor for these peaks in the TIC spectrum, tk le S/N is greatly improved by using the SIC mode.
The use of the SIC mode allows us to unambiguously massidentify each digest fragment in the chromatogram. In these experiments, we simultaneously collect, average, and store the mass spectrum several times per second for the entire chromatogram, so that a complete record of the ions stored in the trap and detected by the reTOF is obtained. The mass spectra corre sponding to the SIC peaks of Figure 3 are shown in Figure 5. In Figure 5a, it is demonstrated that the peak in the ion chromatogram is actually composed of two masses which cannot be resolved by the chromatographic separation but which can easily be identified by the use of mass spectrometry. In addition, it should be noted that there are no solvent clusters or other interfering background peaks at low-mass. In the ion trap, the low mass cutoff can be set to eliminate these background ions so that they are not stored in the trap and detected. It should also be noted that there are no major cluster peaks observed in the mass spectrum. The long storage times and high pressure of buffer gas in the trap provide a mechanism for collisional dissociation of these clusters. The resolution of the IT/reTOFMS in these experiments is between 2500 and 3000. However, the actual resolution recorded in the spectra of Figures 5 and 8 is only -1500. This is because the data acquisition was performed using a time resolution of 10 ns instead of the 5 ns required to observe the full resolution. This was done to limit the data file size, to speed the data transfer process between the transient recorder and the PC memory, and to observe the mass spectra and TIC on the video monitor in real-time. In these experiments, we are collecting and storing the mass spectra over the entire time period of the chromatogram, which may be on the order of 30-50 min. Our software has been developed to signal-average and store the entire mass spectrum, where a mass spectrum is recorded every 2 s. Over the course of the chromatographicseparation, there may be as many as 1500 mass spectra actually stored in the file. Nevertheless, at the end of the chromatographic run, the total data collected can be reprocessed by our software to produce a 3D plot of the separation as a function of mass (not shown here). Although of limited utility in these particular examples, the ability to generate such 3D information may be important in separation of complex unknown enzymatic digests or identification of protein modifcations where the information can be displayed in a manner very similar to that of 2D electrophoretic gel separations often used in biological studies of proteins or proteolytic digests. For example, if a known protein was modified, then upon digestion and analysis by chromatography/ESI/lT/reTOF device, the modified digest fragment would clearly be identifiable as a shifted spot on a 3D image. To further demonstrate the fast data acquisition capability of the LC/IT/reTOF system, we performed an LC/MS analysis of bovine b a s e i n tryptic digest. In Figure 6 are shown the UV trace at 214 nm and the full-scan TIC profile of the tryptic digest obtained with the microbore column separation from an injection of 20 pmol of Bcasein in an injection volume of 5 pL. The TIC intensity and peak profiles of the peptide are qualitatively similar to those of the UV trace. The mass spectrometer was operated under identical conditions as for the cytochrome c tryptic digest separation. No baseline correction or peak smoothingtechniques were used for the chromatogram of Figure 6b. The separation was performed using a solvent gradient from 0%B to 80%B over a period of 60 min. Under these conditions, the total chromatogram required 50 min. Analytical Chemistty, Vol. 67, No. 17, September 7, 1995
2875
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Figure 6. (a) UV trace at 214 nm and (b) total ion chromatogram of the separation of 20-pmol tryptic digest of bovine @-casein.
Bovine /%caseinis a relatively large protein, with 209 amino acid residues and an average mass of 23 583.4 amu. Among the 16 possible tryptic fragments and their combinations, at least 15 fragments have masses higher than 1500units. Under the current ion trap storage conditions during the LC/MS separation, these fragments will not be readily detected if multiply-charged ion species are not formed to a detectable level. Although not all components are completely resolved under the experimental conditions, as observed from both the UV trace and the TIC profile, at least 25 peaks were detected in the TIC profile. The selected ion chromatogram profiles can be used to determine the identity of the peaks in the TIC profile. Figure 7, parts a-c, shows the SIC profiles at m/z 831.4, 781.5, and 569.4, respectively. One important observation from these SIC profiles is that the relatively fast acquisition speed may aid the peak interpretation process by differentiating closely eluting fractions. For example, from the relevant SIC profiles, one can conclude that the unresolved peak eluted at -27.7 min in the TIC contains two closely eluting peaks. The SIC also shows that one of them is further composed of two coeluting peaks with different molecular weights. The elution time difference between these two sets of peaks is only -6 s and would not be differentiated at slower acquisition speed. However, using the SIC mode, it is possible to monitor the identities of each peak. The accompanying mass spectra are shown in Figure 8, where Figure 8a shows the mass spectrum of the fraction eluted at -27.6 min, whereas Figure 8b shows the mass spectrum of the two coeluting fractions, labeled as M and M respectively, with a retention time of -27.7 min. Some of the identified fractions of the bovine B-casein tryptic digest with both measured and calculated masses'are listed in Table 2. 2876
Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
25
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Retention Time (min)
Figure 7. Selected ion chromatograms of bovine 3 @-caseintryptic peptides from 20-pmol injection with center masses at (a) m/z831.4. (b) m/z 781.5, and (c) m/z 569.4. 100
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CONCLUSION
The ion trap storage/time-of-flight mass spectrc )meter serves as a rapid method of detecting the eluent of a liqt lid chromatographic separation over a broad mass range with a hi: gh duty cycle.
Table 2. Comparison of Calculated and Measured Tryptic Fragments of Bovine p-Casein from the LClMS Analysis
no.
fragment
calcd mass
measd massu
sequence
3 3, 4 4, 5 8.9.10 , , 9 10,ll 11 13 14 16
26-28 26-29 29-32 98-107 100-105 106-113 108-113 170-176 177-183 203-209
374.24 502.34 517.34 1139.40 646.32 1013.52 748.37 781.48 830.45 742.45
374.0 502.2 516.9 1137.9 646.5 1013.7 748.2 781.5 831.4 742.5
INK INKK KIEK VKEAMAPKHK EAMAPK HKEMPFPK EMPFPR VLPWEK AWYPQR GPFPIW
Average mass of all charge states of the fragment observed.
In the case of microbore HPLC using an electrospray ionization source, total ion chromatograms of tryptic digests of proteins and peptides such as cytochrome c and bovine ,&casein can be routinely obtained in the 2Gpmol regime. TIC detection can even be obtained down to the 1-pmol level with some loss of chromatcgraphic information. With use of software developed in our laboratory,the data can be reanalyzed to provide the selected ion chromatograms for each chromatographic peak. The SIC mode provides further enhancement of the S/N, so that subpicomole
detection can be obtained for each component. In addition, the SIC mode allows identification of a chromatographic peak with a particular mass. Further, the resolution of the mass spectra provides the ability todetect components that cannot be resolved by the chromatographic separation. Using this methodology, the tryptic fragments of cytochrome c and Pcasein could be separated and mass identified with excellent sensitivity in a relatively simple and inexpensive device. ACKNOWLEDGMENT We would like to thank Michael Lang and Robert Stetler of Varian for the loan of software and other equipment. We would also like to thank Dr. S. E. Buttrill, Jr., for a critical reading of the manuscript. The work described herein received support under several sources which we wish to gratefully acknowledge,including the National Science Foundation, Biological Instrumentation and Instrumental Develop program under Grant No. BIR-9223677, the National Institutes of Health under Grant No. lROl GM495W OM,and Varian Associates, Inc., Ginzton Research Center. Received for review February 23, 1995. Accepted June 12,1995.@ AC9501957 @Abstractpublished in Advance ACS Abstracts, July 15, 1995.
Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
2877
