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he time-of-flight (TOF) mass spectrometer has become a widely used tool for mass analysis of large biomolecules (1). It can rapidly analyze an entire mass distribution simultaneously,which is particularly important when the ionization source has a low duty cycle. The instrument's greatest advantage, however, is that it is a relatively simple mass analyzer, with no moving parts, scanning electric fields, or slits. It is inexpensive, easy to build and maintain, and can analyze large ions with reasonable resolution and mass accuracy. The quadrupole ion trap is a powerful tool for mass analysis and storage of ions over a wide mass range with excellent detection limits. It has been used with many ionization sources, including electron impact, chemical ionization, photoionization, and matrix-assisted laser desorption/ionization (MALDI) (2),which
Mark G. Qian David M. Lubman The University of Michigan
A hybrid instrz&ment that combines TOF with the ion trap yields excellent sensitivity for small samples
could allow exploitation of the advantages of TOF and the ion trap is a combination quadrupole ion trap (IT)/reflectron (re) TOF mass spectrometer (5).Ions stored in the trap could be ejected using the pulsed dc mode into the reTOF half of the instrument for mass analysis.This configuration results in a hybrid nonscanning mass spectrometer that combines the selective storage and MS/MS capabilities of the ion trap with the speed, resolution, and high mass capabilities of the reflectron TOF spectrometer. The capabilities for long-term storage and ejection of can create ions directly inside the trap. In unwanted background can provide exceladdition, techniques that require the introduction of ions from an external source, lent sensitivityfor small samples. In addition, the storage capabilities of the trap such as atmospheric pressure sampling provide a convenient way to interface a glow discharges and electrospray (3), have been interfaced to ion traps. The trap low-intensitycontinuous ion source such as electrospray to a reTOF spectrometer. also has been used to store and analyze high-mass ions and recently has been used This configuration allows detection of electrospray ionization (ESI)- produced ions to achieve extraordinarily high resoluover a wide mass range in a nonscanning tion (4). However, the key feature of the ion trap is its ability to obtain high sensitiv- mass spectrometer so that rapid analysis of chromatographic eluents can be ity through ion storage and integration of achieved. The trap also can operate at elethe signal over an extended period. vated pressures to cool MALDI-proOne instrumental codguration that
234 A Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
0003- 2700/95/0367-234A/$09.OO/O 0 1995 American Chemical Society
duced ions and provide for enhanced resolution and extensive fragmentation; this allows structural analysis of MALDIactivated ions by long-term storage in the trap. In addition, there is the possibility of developing a compact, simple version of this instrument that may find use in biomedical studies and the biotechnology industry. In this Report, we will describe the instrument built in our laboratory and demonstrate its usefulness in a variety of a p plications. Background
TOF mass spectrometers can be used with ion reflectors to obtain excellent resolution for biomolecules in the mass range < 3000 u (6);they can be complemented with jet cooling (7, 8),post-source pulse focusing (9),and delayed extraction methods (IO),which have been used to obtain resolution in excess of several thousand using various ionization sources. TOF instruments do have a major limitation in that they cannot do selective ion storage before mass analysis. Ion storage is important for trace analysis via ion integration using trapping methods (3,11, 12).In addition, the ability to selectively store target ions in ion traps and eject unwanted background ions is important for improving the spectrum’s S/N and ultimately as a means of selecting ions for structural analysis by MS/MS. Although there have been several attempts either to store or to selectively eject ions in TOF instruments by using dc fields, success has been limited. A number of TOF configurations have been developed, however, to obtain structural information, including a tandem TOF instrument to achieve MS/MS (13).An alternative instrument for achieving both ion storage and tandem MS is the ion trap mass spectrometer. In the ion trap mass spectrometer, se-
lective ejection and storage of target ions can be achieved by applying various auxiliary rf fields to the end caps of the trap in a technique called axial modulation. This has led to the unique ability to perform multiple stages of tandem MS in combination with collision or photodissociation fragmentation techniques inside the trap (14).This is unlike tandem MS using a triple quadrupole, where multiple MS stages are required, with transmission limitations due to scanning the multiple quads in tandem. Although tandem MS can be done with FT-ion cyclotron resonance instruments, the trap can operate at the high
The reflectron compensates for the dijjfireme iut the TOF of ions with dijjfirent energies to focus them at the detector pressure torr) critical for effective collision-induceddissociation (CID) and for interfacing to GC and LC. The quadrupole ion trap does have several disadvantages. Although it can store high-mass ions, when it is used in the mass-selectiveinstability mode (used for ejecting ions out of the trap as a function of mass) it may be difficult to scan the rf voltage at a sufficiently large value to scan out high-mass ions. A number of other scan modes, such as axial modulation (1I), can be used to scan high-mass ions out of the trap. The drawback of using this method, however, is that some of the ac-
curacy of the mass calibration is lost. In addition, very high resolution (> 100,000) has been achieved in the trap by scanning the rf voltage very slowly (4). However, the rate at which the mass range is scanned to achieve this high resolution is impractical for many applications, and the resolution and mass accuracy will be affected by the density of ions in the trap and the effects of space charge as the density increases. Although various scan methods have been developed to correct for some of the drawbacks of scanning quadrupole ion traps, an alternative detection method is to use pulsed dc ejection to move the ions from the trap to the detector. The dc pulse is applied to the exit end cap, destabilizing the trap and ejecting the trap contents for analysis. Pulsed dc ejection has been used for more than 20 years to interface ion traps to quadrupole and sector mass spectrometers (15-17). Some of these studies have focused on the fundamental aspects of dc ejection whereas others have used TOF analysis to study the energy distribution and mean kinetic energy of the ion cloud in the trap. More recent hybrid instruments have ion traps interfaced to other ion traps and hybrid BEQ-type devices (17). lnstrumentation Figure 1is a schematic of an ESI source interfaced to an IT/reTOF instrument (5). Here the ion trap has replaced the acceleration region of a standard Mamyrin reflectron source (R. M. Jordan Co.). The key to the operation of the IT/reTOF spectrometer is that ions stored in the trap are not scanned out sequentially, as in the usual ITMS configuration. Instead, they are pulsed out of the trap and into the reTOF instrument for analysis. The ions are ejected by placing a dc pulse on an end cap. Following ejection,
Analytical Chemistry, Vol. 67, No. 7, April I , 1995 235 A
Ion reflector
Skimmer
v
extr
I
---I
I .
Einzel lens
I_
4” Diffusion
Pump
I
.Om
Figure 1. Schematic of the IT/reTOF mass spectrometer with an ESI source. is the interface focusing lens voltage; V, and V, are pretrap Einzel lens potentials; V, is the rf potential; Vextris the dc extraction voltage; V, = which is the flight tube liner voltage; ,V,, is the focusing voltage; ,V, is the beam steering voltage; and V, and ,V, are ion reflector voltages.
,V ,,, V,,
they are accelerated into the reTOF drift tube using an acceleration grid, followed by a drift tube liner inside the flight tube, which is maintained at an elevated voltage. The ions are reflected by the ion reflector and detected at a high-gain triple microchannel plate detector. The trap is cross-drilled through the ring electrode and the end caps. Holes in the end caps allow introduction of external ions from the electrospray interface into the trap as well as ejection of ions from the trap to a TOF detector on the other end. Two holes in opposite ends of the ring electrode allow a laser source to pass through the trap for photoionization or MALDI experiments. ESI or MALDI can be performed in the same instrument without further modification. A standard quadrupole ion trap used in several commercial instruments is incorporated into the instrument. It consists of two end cap electrodes with hyperbolic surfaces and a ring electrode situated between the end caps. A buffer gas can be added to the trap so that it typically operates at 5 x IO4 to torr. This gas is critical for storage of externally injected electrospray-produced ions and hot ions produced in the MALDI process. During operation the trap is held at 0 V dc with an rfvoltage of 1.1MHz; the amplitude a p plied to the ring electrode varies between 0 and 5000 Vpp(peak-to-peak rf voltage). The rf frequency of 1.1MHz used in these experiments is the value often used in
traditional ion trap work. However, much lower frequencies (75-350 kHz) have been used with the IT/reTOF instrument to extend the upper mass range of the trap without further modificationsto the apparatus. The trapped ions can be stored for periods from 10 ps to > 10 s. The repetition rate of the entire experimental cycle varies, depending on the storage time of the experiment. However, the storage time can be easily varied in relation to the number of ions injected into the trap and the degree of ion storage and sensitivity required. The ability to change the storage time is important in controlling the ion density in the trap and thus preventing space charge effects that may limit the resolution in the reTOF instrument. In addition, the storage time can be varied to follow the time evolution of the signal in MALDI activation/fragrnentation experiments or in ion-molecule reactions. Instrument performance
ESI has become a powerful tool for ionizing high molecular weight and fragile biological molecules for analysis by MS (18).A key advantage of this source is the production of multiplycharged ions that allow higher molecular weight samples to be detected in a mass spectrometer with a limited mass range. Thus electrospray has become widely used as an ionization source for biomolecular analysis, using relatively inexpensive quadrupole
230 A Analytical Chemistry, Vol. 67, No. 7,April 1, 1995
mass spectrometers and, more recently, ion traps. Another important characteristic of ESI is that it allows production of ions from species dissolved in solution, making it a widely used method for online interfacing of LC to MS. The IT/reTOF instrument presents a means of interfacing ESI to TOF detection with high sensitivitybecause of the high duty cycle available through the long storage times and the detection speed of this nonscanning device (19).The high duty cycle and storage capability is critical in ESI, which produces continuous ion beams of low ion intensity. The long-term storage in the trap permits integration of the ion signal over a sufficient period to allow detection of relatively strong signals from a weak ion beam. For example, if ions are collected and stored over a period of 50 ms, and the time for ion ejection and detection is only 100 ps, the duty cycle is 99.8%.Ion storage times can be extended to seconds if necessary to measure signals from ultralow concentrations of sample. The storage time in each case will ultimately be limited by the ion density in the trap, which may result in space charge effects and loss of resolution in the IT/ reTOF instrument if the ion density becomes too high. The main problem in interfacing ESI to TOF MS is that ESI produces a lowintensity and continuous ion beam in contrast to TOF analysis, which requires pulsed operation to achieve time resolu-
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Analytical Chemistry, Vol. 67, No. 7, April 1, 1995 237 A
before the ions are pulsed into the reTOF spectrometer for detection. Flow injection is used to introduce the sample so that it can be signal averaged over multiple spectra to enhance S/N. The actual amount of sample consumed in obtaining this spectrum is 22 pmol. The isotopic distribution of the molecular ion (m/z 712) is clearly resolved down to the baseline with excellent S/N. The fwhm of this peak is < 20 ns, which corresponds to a mass resolution approaching3000. An actual measurement of the sensitivity of this method corresponds to detection limits of nearly 4.5 and 0.5 fmol for gramicidin S and arginine, respectively. Several methods are used to induce fragmentation in molecules, such as CID of electrospray-produced ions of peptides between the capillary exit and the skimN
Figure 2. ESI spectrum of leuenkephalin-arg showing the expanded view of the molecular ion region.
mer of the electrospray interface (23).By varying the voltage on the focusing ring inside the vacuum interface, the ions can be accelerated to produce extensive fragmentation, as illustrated in Figure 3. In this ESI/IT/reTOF CID mass spectrum of melittin obtained using an accelerating voltage of 280 V (19),the fragmentation spectrum is produced from all the different multiply-chargedions simultaneously entering the ESI interface. The CID spectrum is quite similar to that published by Smith et al. (23),who used a quadrupole spectrometer to obtain their results. Although the fragmentation appears quite extensive, it resembles low-energy CID mass spectra such as those in Reference 25. The CID spectrum provides an extensive set of b and y ions that can be used for structural analysis, as shown in Figure 3.
tion. Other configurations have been used to interface ESI to TOF MS, most notably orthogonal pulsed extraction of ions produced from an ESI source into a TOF instrument, which generates discrete ion packets with a sufficiently narrow time profile to provide high TOF resolution (20-22). Although high resolution has been achieved with this method, it is inherently a low duty cycle instrument because it extracts ions only over a very short period. To achieve a high duty cycle in this experiment, a pulsed extraction rate > 2000/s must be used. This high repetition rate, along with the potentially large record lengths and speed with which the acquired spectra must be stored, requires specially designed circuitry and software or may require ion counting techniques. In the IT/reTOF mass spectrometer, the ion trap serves to convert a lowa5- 17 intensity continuousbeam into a higher intensity pulsed source with a high duty cydbA-17 cle but a low pulse-out extraction rate. This low pulseout rate can, in turn, simplify the required electronics and data processing and allow conversion of a lowintensity ion beam into a signal of sufficient intensity to permit the use of analog electronics. In addition, the use of the trap interface can provide selective storage of target ions with ejection of unwanted ions and solvent background. The ion trap also provides the capability to use MS/MS for structural analyses not easily performed with other configurations. Figure 2 shows the ESI mass spectrum of the expanded molecular ion (MH') region for a 3 x M solution of leuenkephalin-arg. The spectrum is obtained using a trap storage time of 931 ms Figure 3. ESI CID spectrum of melittin.
I
238 A Analytical Chemistry, Vol. 67, No. 7, April I , 1995
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Analytical Chemistty, Vol. 67, No. 7, April 1, 1995 239 A
Repoff/
(M+3H)3+(949.5)
I
Figure 4. Expansionof Figure 3 showing isotopic distribution of the (M + 3H)=+,yle2+, and b,, ions.
A fairly large number of fragments also are observed that were not seen by Smith et al. (23);many fragments below m/z 600 were seen in our work. The striking feature of the CID spectrum shown in Figure 3 is that the entire spectrum over a broad mass range is simultaneously stored and integrated over an extended time (931 ms) from the lowintensity ESI source and then simultaneously detected by the reTOF mass spectrometer. Thus, the duty cycle for detection over an almost 1500 amu mass range in the IT/reTOF is nearly 100%. Note that little signal is observed below m/z 70 because of the low mass cutoff of the ion trap at the set rf voltage. This may be an advantage because background solvent ions from the ESI source are eliminated from the mass spectrum. The ability of the IT/reTOF instrument to resolve ions is also demonstrated in Figure 4, which shows an expanded region of the (M+3H)3+ion of the spectrum in Figure 3. Because the ion peak is triply charged, the isotopic spacing is onethird that of the singly charged peak. Nevertheless, the isotopic distribution of the (M+ 3W3+ion is clearly resolved. In comparison, the singly charged b,, peak and the doubly charged yls peaks illustrate the difference in the isotopic spacing, which depends on the charge. The spectra in Figures 3 and 4 were obtained at a flow rate of 0.5 pL/min at a sample concentration of 6 x M, corresponding to
-
a total sample consumption of 48 pmol. In the IT/reTOF configuration, the trap serves as a means of performing MS/MS for TOF MS analysis. This can be done using low rf voltage (- 0.5 V) on one of the end caps to eject unwanted ions, leaving only the target ions in the trap. A second rf voltage ( 0.2 V) at the characteristic resonance frequencies of the target ions is used to accelerate them into the helium buffer gas in the trap to induce CID. The ions are then pulsed into the reTOF spectrometer for detection. This method allows mass selection of an ion before CID and provides structural information, as well. In these MS/MS experiments for electrospray-produced ions, such as bradykinin and leu-enkephalin-arg, TOF provides a convenient method for detection of relatively large ions and their fragments.
-
Peptide digest analysis
The nonscanning character of the IT/reTOF instrument allows unique on-line detection of chromatographic separations because of its ability to quickly obtain a spectrum over a broad mass range with high resolution. Such an approach is useful in capillary separations, where the peak width may be on the order of several seconds. An ESI spectrum can be obtained with storage times of < 30 ms without experiencing significant loss of resolution, so that sampling rates of at least 30 per second can be achieved. This speed is sufficient for even the fastest of separations.
240 A Analytical Chemistry, Vol. 67, No. 7,April 1, 1995
The sensitivityand resolution of ESI/ IT/reTOF MS is of particular interest in solving biological problems requiring chromatographic separation and on-line analysis. One problem under study in our laboratory is the sequencing of small amounts of proteins extracted from tumor cells and separated on 2D gels. Often there may be only picomole or subpicomole quantities of the proteins available followinggel separation. After extraction from the gels, the proteins are generally enzymatically digested and separated by microbore and capillary LC methods. The sensitivityand speed of the IT/reTOF instrument are critical for detecting the narrow peaks eluting from the column. The resolution and mass accuracy available from the instrument are also important for identification of tryptic fragments, especially when modifications such as phosphorylation are present. Figure 5a shows a chromatogram of a microbore HPLC separation of the tryptic digest of 100 pmol bovine cytochrome c. The separation was performed on an Alltec C,, microbore column (1.0 mm i.d.) using an acetonitrile/water gradient elution at a flow rate of 50 pL/min. Detection was achieved by monitoring UV absorption at 214 nm from a 60 nL microcapillary cell. For comparison, the eluent from the UV detector was then directly passed to the ESI source, ionized, and injected into the IT/reTOF spectrometer for detection by mass analysis (Figure 5b). This total ion chromatogram (TIC) mode of detection, which results in a chromatogram qualitatively similar to Figure 5a, was obtained by monitoring the total ion current over the mass range 60-150011. There are some differences in the relative peak heights in Figures 5a and 5b, attributable to the relative differences between UV a b sorption detection efficiency and the d e tection of ion peaks by mass analysis. The nonscanning capabilities allow the TIC data to be obtained rapidly and at detection levels as low as 4 pmol. In addition, the data can be processed to provide a selected ion chromatogram (SIC) for each mass detected, as shown in Figure 5c for the peak at 25 min from Figure 5b. This method allows correlation of each chromatographic peak with one or more particular masses. Because the background ions have been eliminated from
the integrated Chromatogram,the S/N is greatly improved. A detection limit for online analysis of a cytochrome c digest of 1pmol is routinely obtained using this method (which is limited by the presence of organic modhers in the mobile phase). We can also 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 spectrometer is obtained. The corresponding mass spectrum of the SIC
of Figure 5c is shown in Figure 5d for m/z 1307 and 634, where two masses are unresolved in the chromatographic peak.
N
MALDI MS
MALDI is a powerful method for generating ions of large molecules for MS analysis (1-3). Generally, MALDI is performed in linear TOF devices, although some work on MALDI using reTOF and ITMS has been done recently. Ion trapping has several unique advantages for MALDI MS in combination with TOF analysis when
researchers must examine very small samples. In principle, the IT/reTOF spectrometer can provide very high sensitivity in MALDI, where ions from multiple laser pulses can be stored and the signal effectively integrated while the matrix background at low mass is eliminated by the low mass cut-off or resonance ejection techniques. A common problem encountered in MALDI experiments is low resolution attributable to the energy spread following desorption or metastable decay. The use to 10-4torr in the of a buffer gas at trap can effectivelycool the large hot ions produced by the desorption process, resulting in much-improved resolution. This improved resolution was demonstrated when peptide ions were produced by MALDI from a metal insertion probe directly inside the trap (24). A single laser pulse of MALDI-produced ions was stored for 90 ms to generate a very limited number of ions, resulting in resolution of up to 6000, where the MH' ion peak and its isotopic counterparts were resolved down to baseline. However, this level of resolution can be obtained only under tightly controlled conditions of ion density in the trap. As the number of ions in the trap increases, space charge effects will dominate and limit the resolution. Nevertheless, a resolution of 1000 can be routinely obtained in these experiments. A second problem in MALDI MS experiments is the lack of fragmentation to provide structural information. An important recent breakthrough is the ability to obtain extensive sequence information for peptides in MALDI by using the IT/reTOF spectrometer to extend trap ping times, allowing long-lived decay to occur in the trap (25).The results obtained are similar to those observed by post-source decay experiments in reTOF MS (26). However, the post-source decay in these experiments was performed in a reTOF spectrometer with a limited flight time ( 100 us), where the decay was incomplete. We found that in the IT/ reTOF instrument, ion decay can be observed even after 10 ms. The decay time depends on the internal energy placed in the ion during the MALDI process and appears to be sensitive to the laser N
N
Figure 5. LCMS chromatogramof 100 pmol of bovine cytochrome c tryptic digest. (a) UV spectrum at 214 nm, (b) TIC in a mass window from 60 to 1500 u, (c) SIC at m/z 634, and (d) mass spectrum of the peak shown in (c).
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995 241 A
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power and wavelength, matrix, and rf voltage on the ring electrode. We found this to be the case for the peptide angiotensin 111. The spectrum was obtained followingMALDI activation at 355 nm and a trap storage time of 50 ms so that decay was complete. The laser power, trapping, and ejection conditions were optimized for ion fragmentation at the expense of resolution. The ion decay provides a nearly complete set of b and b,, peaks for sequencing under these conditions. Similar results have been obtained for a number of peptides including substance P, neurotensin, angiotensin I, bradykinin, and melittin. In most cases a relatively complete set of b or y fragments is observed. However, it is significant that the decay is completed within the trap so that the long-lived decay is observed as stable peaks in one complete spectrum from the reTOF spectrometer. In postsource decay reTOF experiments the decay is detected as metastable ions and the reflectron energy analyzer must be scanned over an extended period to obtain a complete spectrum. Outlook
There are currently 15-20 IT/reTOF spectrometers of varying designs in laboratories around the world. Although the published literature still remains sparse on this relatively new mass spectrometric instrument, we expect that the need for fast response, high sensitivity,and structural analysis in chromatographic detection will make the IT/reTOF spectrometer the analyst's instrument of choice in such applications. We thank Michael Lang and Robert Stetler (for the loan of software and other equipment) and S. E. Buttrill, Jr. (for a critical reading of the manuscript) of Varian. We gratefully acknowledge the National Science Foundation, the National Center for Human Genome Research, and Varian Associates, Inc., Ginzton Research Center.
References (1) Cotter, R. J.Anal. Chem. 1992,64,1027 A-1039 A. (2) Chambers, D. M.; Goeringer, D. E.; McLuckey, S. k;Glish, G. L. Anal. Chem. 1993,65,14-20. (3) McLuckey, S. A; Van Berkel, G. J.; Goeringer, D. E.; Glish, G. L. Anal. Chem. 1994,66,689 A-696 A. (4) Cooks, R G.; Williams, J. D.; Cox, K. A;
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Kaiser, R. E., Jr.;Schwartz, J . C. Rapid Comm. Mass Spectrom. 1991,5,327-29. (5) Michael, S. M.; Chien, B. M.; Lubman, D. M. Anal. Chem. 1993,65,2614-20. (6) Boesl, U.; Neusser, H. J.; Weinkauf, R.; Schlag, E. W.J. Phys. Chem. 1982,86, 4857-63. (7) Lubman, D. M.; Jordan, R M. Rev. Sci. Znstrum. 1985,56,373-76. (8) Opsal, R B.; Owens, K. G.; Reilly, J. P. Anal. Chem. 1985,57,1884-89. (9) Kinsel, G. R; Grundwuermer, J. M.; Grotemeyer, J. J. Am. SOC.Mass Spectrom. 1993,4,2-10. (10) Colby, S. M.; King, T. B.; Reilly, J. P. Rapid Comm. Mass Spectrom. 1994,8,865-68. (11) Cooks, R G.; Kaiser, R E., Jr. Acc. Chem. Res. 1990,23,213-19. (12) Nourse, B. D.; Cooks, R G. Anal. Chem. Acta 1990,228,l-21. (13) Cornish, T. J.; Cotter, R J. Rapid Comm. Mass Spectrom. 1993, 7,1037-40. (14) Cooks, R J.; Glish, G. L.; McLuckey, S. A; Kaiser, R E., Jr. Chem. Eng. News, March 25, 1991, pp. 26-41. (15) Mosburg, E. R.; Vedel, M.; Zeraga, Y.; Vedel, F.; Andre, J. Znt. J. Mass Spectrom. Zon Proc. 1987, 77,l-12. (16) Lifshitz, C.; Nadav, E.; Peres, M.; Peres, T.; Laskin, J.; Karsenty, B.; Shaked, M. Znt. J. Mass Spectrom. Zon Proc. 1994,133, L1l-Ll4. (17) Suter, M.J.F.; Gfeller, H.; Schlunegger, U. P. Rapid Comm. Mass Spectrom. 1989, 3,62-66. (18) Fenn, J . B.; Mann, M.; Meng, C. IC;Wong, S. F.; Whitehouse, C. M. Science 1989, 246,64-71. (19) Chien, B. M.; Lubman, D. M. Anal. Chem. 1994,66,1630-36. (20) Boyle, J. G.; Whitehouse, C. M. Anal. Chem. 1992,64,2084-89. (21) Mirgorodskaya, 0. A; Shevchenko, A. A.; Chemushevich, I. V.; Dodonov, A. F.; Miroshnikov, A. I. Anal. Chem. 1994, 66, 99-107. (22) Verentchikov, A. N.; Ens, W.; Standing, K. G. Anal. Chem. 1994,66,126-33. (23) Smith, R D.; Loo, J . A; Barinaga, C. J . ; Edmonds, C. G.; Udseth, H. RJ. Am. SOC. Mass Spectrom. 1990, I , 54-65. (24) Chien, B. M.; Michael, S. M.; Lubman, D. M. Rapid Comm. Mass Spectrom. 1993, 7,83743. (25) Fountain, S. T.; Lee, H.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1994,8, 407-16. (26) Spengler, B.; Kirsch, D.; Kaufmann, R. J. Phys. Chem. 1992,96,9678-84.
David Lubman's research interests include protein and DNA sequencing, instrument design, and bacterial screening. Mark Qian's research interests focus on LC/MS instrumentation, high-resolution electrophoresis, and separation and characterization of proteins and low molecular weight compounds of biomedical interest. Address correspondence about this article to Lubman at the Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109.
