Art ides Anal. Chem. 1994,66, 1601-1609
Aerosol Matrix-Assisted Laser Desorption Ionization for Liquid Chromatography/Time-of-Flight Mass Spectrometry Kermit K. Murray, Tanya M. Lewis, Michelle D. Beeson, and Davld H. Russell' Department of Chemistry, Texas A&M University, College Station, Texas 77843-3255
We report the application of aerosol matrix-assisted laser desorption ionization (MALDI) to liquid chromatography/ mass spectrometry (LC/MS). The aerosol MALDI experiment uses aerosol liquid introduction in conjunction with pulsed UV laser ionization to form ions from large biomolecules in solution. Mass analysis is achieved in a time-of-flight mass spectrometer. In the LC/MALDI-MS experiment, the matrix solution is combined with the column effluent in a mixing tee. LC/MALDI-MS is demonstrated for the separation of bradykinin, gramicidin S, and myoglobin. There is an ongoing effort in analytical mass spectrometry to develop liquid chromatography/mass spectrometry (LC/ MS) techniques that approach the analytical utility of GC/ MS.1-3 The principle advantage of gas- or liquid-phase separation followed by mass spectrometric detection is that analyte molecules are identified both by chromatographic retention time and by molecular weight and fragmentation pattern. Two-dimensional (2D)separation by chromatography and M S is particularly important for the analysis of complex biological and environmental samples. The inherent difficulty in developing an LC/MS interface is the incompatibility of liquid introduction with the high vacuum requirements of a mass spectrometer. For example, 1 cm3 of water will expand to a volume of lo5 m3 when introduced into a mass spectrometer at Torr. Therefore, solvent and analyte must be separated before introduction into the mass spectrometer. The most successful and commonly used LC/MS techniques are based on aerosol liquid introduction. Aerosols promote rapid solvent evaporation due to their high surface-to-volume ratio.4 The 1 cm3 of water (1) Arpino, P. J.; Guiochon, G . Anal. Chem. 1979, 51, 682A-701A.
(2) Covey, T. R.; Lee, E. D.; Bruins, A. P.; Henion, J. D. Anal. Chem. 1986.58, 145 1A-1 46 1A. ( 3 ) Huang, E. C.; Wachs, T.; Conby, J. J.; Henion, J. D. Anal. Chem. 1990,62, 713A-725A. (4) Browner, R. F. Microchem. J. 1989, 40, 4-29.
0003-2700/94/0386-1601$04.50/0 0 1994 American Chemical Society
has a total surface area of 6000 cmz when nebulized as 10pm-diameter aerosol particles. Aerosol LC/MS techniques may be classified by both the mode of aerosol formation and method of ionization. The common methods of aerosol formation for LC/MS are pneumatic, ultrasonic, thermal, electrostatic potential, or some combination. In some cases, ion formation occurs as a direct consequence of nebulization: the nebulized droplets are charged and solvent evaporation leads to droplet breakup and the ejection of ions. The electrospray technique is used to produce charged droplets in a corona discharge.5.6 Electrospray is limited to flow rates below 40 pL/min and thus cannot be used at conventional high-performance liquid chromatography (HPLC) flow rates without postcolumn splitting of the f l o ~ . Several ~ , ~ variations of electrospray have been developed utilizing either pneumatic7 or a combination of pneumatic and ultrasonic nebulization8 to assist droplet formation. The assisted electrospray techniques can be used at flow rates in excess of 1 mL/min without postcolumn splitting. The thermospray technique uses a heated capillary to produce charged droplet^;^ however, it is often used in conjunction with electron impact or corona discharge ionization.2 Although thermospray is compatible with HPLC flow rates, it is limited by the need for careful temperature control and a widevariation in sensitivity for different analytes2 Pneumatic nebulization has been used in conjunction with either chemical or electron impact ionization.10 As improved LC/MS interfaces and ionization methods evolve, the compatibility of the mass analyzer becomes an L.L.; Hines, R. L.;Mobley, R. C.; Ferguson, L.D.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (6) Fenn, J. B.; Mann, M.; Meng, C. K.;Wong, S. F.; Whitehouse, C. M. Mass Spectrom Rev. 1990, 9, 37-70. (7) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987,59,2642-2646. (8) Banks, J. F., Jr.;Shen, S.;Whitehouse, C. M.; Fenn, J. B.Ana1. Chem. 1994, 66,406-414. (9) Blakley, C. R.; Carmody, J. J.; Vestal, M. L. Anal. Chem. 1980, 52, 16361641. (10) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984,56, 2626-2631. ( 5 ) Dole, M.; Mack,
AnaiyticalChemlstry, Vol. 80, No. 10, May 15, 1994 I001
a) Aerosol-Laser Plane
DETECTOR
2400 us 8 10.4 Torr
33ous 8 1 Torr
b) Laser-TOF Plane LASER
A
SOURCE REGION
-
VALVE DIGITAL OSCILLOSCOPE
E ;G
I
(I {
DI~~;;ON
1200 Us 8 10.5 Torr U '386 MICROCOMPUTER Figure 1. (a) Aerosol formation. An aerosol Is produced continuously in the first chamber and sent through a heated tube to the second chamber for ionization.LC/MS is performed by mixing the output of the HPLC with a matrix solution before nebulization. Typical total flow rates are between 0.5 and 1 mL/min. (b) Ionization and timeof-flight mass analysis. A 10-Hz 355-nm Nd:YAG laser creates ions that are accelerated with a lOkV repeller potential for mass separation in a 1.1-m flight tube. Mass spectra are averaged by a digital oscilloscope and sequentially downloaded to a microcomputer during the LC/MS run.
important issue.l13l2 The electrophoretic peak widths in capillary electrophoresis (CE) are typically milliseconds wide and hence place stringent demands on the mass spectrometric data acqui~iti0n.l~ Similarly, fast separations using packed perfusion column HPLC give relatively narrow peak widths.14J5 Consequently, mass analysis methods that are faster and more sensitive must be developed. Although Fourier-transform ion cyclotron resonance (FTICR) and quadrupole ion trap mass spectrometers are attractive as liquid separation mass analyzers,I3J6 the long data acquisition times required, especially for high-mass resolution, limit the compatibility with modern HPLC and CE methods. A number of laboratories are working to develop time-of-flight (TOF) methods for coupling to fast liquid These efforts have focused on (11) Holland, J. F.;Enke,C.G.;Allison,J.;Stults,J.T.;Pinkston, J.D.;Newcome, E.; Watson, J. T. Anal. Chem. 1983, 55, 997A-1012A.
(12) Emary, W. E.; Lys, I.; Cotter, R. J.; Simpson, R.; Hoffman, A. Anal. Chem. 1990, 62, 1319-1324. (13) Smith, R. D.; Wahl, J. H.; Goodlett, D. R.; Hofstadler, S. A. Anal. Chem. 1993, 65, 574A-584A. (14) Kassel, D. B.; Shushan, E.; Sakuma, T.; Salzmann, J.-P. Proc. ASMS Conf: Mass Spectrom. Allied Top., 41st 1993, loa-b. (15) Kassel, D. E.; Luther, M. A.; Willard, D. H.; Fulton, S. P.; Salzmann, J.-P. In Techniques in Protein Chemistry IV, Hogue-Angeletti, R., Ed.; Academic Press: New York, 1993; pp 55-64. (16) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63, 375-383.
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both electrospray and matrix-assisted laser desorption ionization (MALDI). We have recently reported a method for aerosol liquid introduction into a mass spectrometer that uses MALDI.21.22 In the past several years MALDI has become a widely used technique fclr mass spectrometric analysis of biopolymers.23 In a surface MALDI experiment, the analyte biopolymer is deposited from solution onto a metal surface along with a large molar excess of a suitable matrix. The solvent is allowed to evaporate, and the sample is inserted into the source region of a mass spectrometer. Pulsed laser light is absorbed by the matrix, causing both ablation of the surface and ionization of the intact biomolecule by proton transfer.24 The surface deposit and drying steps are eliminated in aerosol MALDI: a solution of matrix and analyte is sprayed directly into the (17) Boyle, J. G.; Whitehouse, C. M. Anal. Chem. 1992, 64, 2084-2089. (18) Verentchikov,A.;Ens,W.;Standing,K.G.Proc.ASMSConJ MassSpectrom. Allied Top., 41st 1993, 4a-b.
(19) Fang, L.; Zhang, R.; Zare, R. N. Proc. ASMS ConJ Mass Spectrom. Allied Top., 41st 1993, 755a-b. (20) Li, L.; Wang, A. P. L.; Coulson, L. D. Anal. Chem. 1993, 65, 493-495. (21) Murray, K. K.; Russell, D. H. Anal. Chem. 1993, 65, 2534-2537. (22) Murray, K. K.; Russell, D. H. J . Am. Soc. Mass Spectrom. 1994, 5, 1-9. (23) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, E. T. Anal. Chem. 1991.63, 1193A-1202A. (24) Gimon, M. E.; Preston, L. M.; Solouki, T.; White, M. A,; Russell, D. H. Org. Mass Spectrom. 1992, 27, 827-830.
mass spectrometer. Following solvent removal and aerosol beam collimation, ions are formed by laser ablation of the airborne particles. Mass selection is achieved in a linear TOFMS. In this article, we report the use of aerosol MALDI as a detector for HPLC separation of peptides and proteins. A preliminary account of LC/MALDI-MS has been reported p r e v i ~ u s l y .Here ~ ~ we report a three-component peptide and protein separation as a demonstration of aerosol MALDI as an LC/MS technique. A mixture of bradykinin, gramicidin S , and myoglobin was separated on a Clg reversed-phase column. After postcolumn addition of matrix, the column effluent was sprayed into the aerosol MALDI mass spectrometer. Data resulting from continuous acquisition yielded a 2D separation by HPLC and aerosol MALDI-MS.
EXPERIMENTAL SECTION The aerosol MALDI apparatus has been described in detail elsewhere2*and will be briefly outlined here. A schematic diagram of the aerosol MALDI TOF-MS is shown in Figure 1. In the aerosol MALDI experiment, the analyte peptide or protein is dissolved in a methanol solution along with an ultraviolet-absorbing matrix. The solution flows into a pneumatic nebulizer at a rate of 0.5-1 mL/min. The aerosol is sprayed into vacuum and desolvated as it passes through a drying tube heated to 500 OC. Ions are formed when the aerosol beam is irradiated by the 355-nm output of a frequencyt r i p M d : Y A G laser operating at 10 Hz. The laser is focused by a cylindrical lens to a 7 mm X 0.2 mm spot at the intersection of the laser and ion beams. The ions are extracted at 10 kV, mass separated in a 1.l-m linear flight tube, and detected with a microchannel plate particle multiplier. Mass spectra are averaged by a LeCroy 9450 digital oscilloscope and downloaded to an IBM compatible '386 microcomputer for analysis. For LC/MS operation, the matrix solution is combined with the HPLC column effluent in a mixing tee. Averaged aerosol MALDI mass spectra are sequentially acquired, downloaded, and stored on the microcomputer. The HPLC system consists of a Waters 600-MS and Model U65 injector with a Clg reversed-phase column, a Waters 484 UV-visible absorbance detector, and a Hewlett-Packard HP3994A integrator. The absorbance detector was set to monitor 214 nm for the experiments described herein. The analog output of the integrator is connected to a digital multimeter interfaced to themicrocomputer through a GPIB connection. The column output is sent first to the absorbance detector and then into a mixing tee. The matrix solution is loaded into a syringe pump connected to the second input arm of the mixing tee and the output is sent to the nebulizer. Mass spectra were acquired and averaged by the oscilloscope and downloaded to the microcomputer via a GPIB interface as successive scans were completed. For the set of experiments described in this paper, the mass spectra were averaged over 100 laser shots. The microcomputer monitored the acquisition process and downloaded and saved the mass spectra to disk. The acquisition and download of a 100 laser shot mass spectrum took an average of 11.4 s to complete, indicating a 12% dead time due to oscilloscope-to-computer K. K.;Russell, D. H.Proc. ASMS Conf. Muss Spectrom. Allied Top., 41sI 1993, 780a-b.
( 2 5 ) Murray,
a) Bradykinin MW = 1060
6 0
1
2
3 mlz (kDa)
4
5
MW = 16,951
0
10
20 30 m/z (kDa)
40
50
Figure 2. Aerosol MALDI mass spectra of (a) bradykinin,(b) gramicidin S, and (c) horse heart myoglobin, each in an a-cyano-4hydroxycinammic acid matrix. The matrix-to-analyte molar ratios were 50 for bradykinin, 6 for gramicldin S, and 90 for myoglobin.
data transfer. Downloaded mass spectra consisted of 20 000 point data files with a time resolution of 10 ns/per point. At the completion of each 10-s scan, the voltage signal corresponding to the 214-nm UV absorption was saved from the digital multimeter to disk. Analyte solutions were prepared by dissolving the analyte in water with 0.1% trifluoroacetic acid. The matrix solution was prepared by dissolving the matrix in methanol and acidifying with 6% trifluoroacetic acid. The compound a-cyano-4-hydroxycinnamic acid (97%; Aldrich, Milwaukee, WI) was used as the matrix. Bradykinin acetate salt (Sigma, St. Louis, MO), gramicidin S hydrochloride from Bacillus brevis (Fluka Biochemika, Ronkonkoma, NY), and horse heart myoglobin (95-100%; Sigma) were used with no further purification. The solvents used were anhydrous 99.8% spectrophotometric grade methanol, spectrophotometric grade acetonitrile (Mallinckrodt, Paris, KY), water produced by a Milli-Q unit (Millipore, Bedford, MA), and trifluoroacetic acid (Sigma).
RESULTS A separation of peptides and proteins was performed to demonstrateaerosol MALDI with LC/MS. Threecommonly used MALDI analytes were used in the separation: bradykinin (MW = 1060), gramicidin S (MW = 1142), and myoglobin (MW = 16 951). A 150-pLinjectionof94nmolofbradykinin, 88 nmol of gramicidin S , and 12 nmol of myoglobin was made onto a Clg column. Two solvents were used in the HPLC separation. Solvent A consisted of water with 1% TFA and solvent B consisted of 90% CH3CN and 10% H2O with 0.1% TFA. The eluent was pumped isocratically at 60% A and 40% B from 0 to 1 min, then ramped linearly to 10% A at 5 min, to 2% A at 15 min, and back to 60% A at 25 min. A total of 196 mass spectra were obtained for the LC/MS run Analytical Chemistry, Vol. 66,No. 10, May 15, 1994
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50
0
150
100 Flight Time (ps)
Flgure 3. LC/MS separation of 94 nmol of bradykinin, 88 nmol of gramicidin S, and 12 nmol of myoglobin on a Cle column. Each mass spectrum in the 3D plot is the 10-s average of 100 laser shots. The solvents were (A) H20 with 1% TFA and (B) 90% CH3CN and 10% H20 with 0.1% TFA. The gradient was 0-1 min 60% A, 40% B, 5 min 10% A, 15 min 2 % A, and 25 min 60% A. Bradykinin appears at a retention time of 12 mln and a flight time of 31 ps, myoglobin at 19 min and 128 ps, and gramicidin S at 30 min and 32 ps.
with each mass spectrum an average of 100 laser shots at a laser pulse energy of 15 mJ (220 MW/cm2). Mass spectra of the three analyte molecules used in the separation, bradykinin, gramicidin S, and myoglobin, are shown in Figure 2. The mass spectra were obtained in direct liquid injection mode with both the matrix and analyte injected into the mass spectrometer in an acidified methanol solution. The matrix used to obtain the mass spectra was a-cyano-4hydroxycinnamic acid. In each mass spectrum, the prominent high-mass peak is associated with the protonated analyte ion, labeled [M + H]+ where M indicates theanalyte. Also present in each mass spectrum in Figure 2 are peaks associated with the protonated analyte cluster ion, labeled [2M + HI+, and the doubly protonated analyte, labeled [M + 2HI2+. In the bradykinin mass spectrum in Figure 2a, adduct peaks can be seen to the high-mass side of the [M H]+ peak. These peaks are assigned as [M Na]+ and protonated bradykinin clustered to the matrix and matrix fragments. The peak associated with [M Na]+can be seen as a high-mass shoulder to the gramicidin S [M + H]+ peak in Figure 2b. Adduct peaks are not resolved in the myoglobin mass spectrum in Figure 2c; however, peak tailing to high mass indicates the presence of adduct ions. In the mass region below approximately m / z 500, the mass spectrum is dominated by protonated methanol clusters,22and in the case of myoglobin, Fe+ clustered to methanol. A three-dimensional representation of the LC/MALDIMS scan for the separation of bradykinin, gramicidin S , and
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myoglobin is shown in Figure 3. The horizontal and vertical axes indicate the ion flight times and the ion signal, respectively. The dimension into the page indicates the LC column retention time. Each mass spectral trace represents the average of 100 laser shots. The bradykinin elutes first with approximately a 12-min retention time. The bradykinin [M + H]+ peak can be seen at 31-ps flight time, just to the right of the low-mass peaks. The myoglobin elutes next, with a retention time of 19 min. The myoglobin [M + H]+peakis seen at 128 ps, and the [M 2HI2+peak is seen at 90 ps. The gramicidin S peak elutes last at a retention time of 30 min, and the [M + H]+ peak appears at a flight time of 32 ps. A color topographic map for the bradykinin, gramicidin S , and myoglobin separation is shown in Figure 4. The data are identical to that shown in Figure 3. The horizontal axis of Figure 4 is ion flight time and the vertical axis is retention time. The flight and retention time scales are identical in Figures 3 and 4. For Figure 4 the number of data points on the flight time axis was reduced to 500; thus the time per data point is 400 ns. The logarithm of the ion signal value for each set of flight and retention times is represented by a color with each color representing a particular range of values. The red areas correspond to the smallest ion signals, whereas theviolet areas correspond to the largest ion signals. The intermediate colors follow the ordering of colors in the electromagnetic spectrum: red, orange, yellow, green, blue, violet. Black indicates that the ion signal is off-scale in the positive direction
+
____I
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. I
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I
100
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Flight Time (ps) /MS separation. The x-axis is ion The bradykinin appears as a do peaks appear at 128- and 90-p
and white indicates that th direction. The areas of the topograp bradykinin, gramicidin S, and identified and are labeled in Figure oriented cigar-shaped areas at a flight ti between retention times of 10 and 18 peak of myoglobin at 19-min retention time in the middle of the plot. The [M H]+ S gives rise to a blue vertically oriented above bradykinin and near the top of the An expanded view of the left half of Figure 5. As in Figure 4, the logarithm used in determining the plot color for e retention times. The color scale has been shift
in the negative
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and eluting
plot. The flight time
+
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Ana&ticaI Ctw&by,
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xis is ion flight time, and the w x i s is HPLC retention the labeled lines are given in Table 1 and discussed
ure 8a. The arrow
rresponds to the elution of the 12.7 min. An indication of gure 8a as the left shoulder t 15-min retention time. A 0 and 34 min is apparent in e difficult to iden me ion signal scale as Figure factor of 20, however, the peaks correspon seen. Ion chromatogramsa data by integrating the
1
grams obtained from the data presented in F along with the 214-nm UV absorption spectr LC run. The integrated regions are number ALDI-MS separation reveals sever 50-ns-wide range of flight
: (1) the bradykinin and to a lesser exten
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20 30 Flight Time (ps)
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10
'
50
40
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100
150
200
Flight Time (ps) Flgure 7. Mass spectra corresponding to the labeled lines of Figure 5. The prominent analyte ions are (a) bradyklnln, (b) ArgPro, (c) bradykinin,(d) gramicldln S (shoulder), (e)gramicidin S, and (9myoglobin. Table 1. Analytes Auoclated wlth the Mass Spectra and Ion Chromatograms Labeled In Flgures 5-8
ma88 spectrum A B C D E F chromatogram
analyte bradykinin Arg-Pro bradykinin gramicidin S (shoulder) gramicidin S myoglobin analyte Arg-Pro bradykinin gramicidin S myoglobin
MALDI ion formation. The appearance of the Arg-Pro [M + H]+ peak in the LC/MALDI-MS spectrum was unexpected because the aerosol MALDI mass spectrum of bradykinin does not show any evidence of this fragment ion. The peaks in Figure 2a can I608
1
~
1
1
1
20
1
~
1
1 1 1 1 11 1~ 1 1 1 1 1 1 1 1 1
Retention Time (min)
30
0
Flgure 8. (a) 214-nm absorptlon spectrum for the LC separatlon. Ion chromatograms obtained from the data shown in Figures 4-6 for (b) bradykinin, (c) ArgPro, (d) gramicldln S, and (e) myoglobin.
all be attributed to intact bradykinin, solvent, matrix, and alkali metal impurities. We attribute the appearance of ArgPro [M H]+ in the LC/MALDI-MS to chemical degradation of bradykinin on or before the LC column (the sequence for bradykinin is Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). Solution-phase degradation of bradykinin is supported by evidence in the 214-nm absorption trace of Figure 8a, where a peak is visible on the left shoulder of the trailing bradykinin peak. This shoulder peak occurs at the 13-min retention time of the Arg-Pro peak in Figure 8b. A closer inspection of Figure 5 reveals the ion corresponding to protonated ProGly-Phe-Ser-Pro-Phe-Arg at a retention time of 16 min and a flight time of 27 ps. The appearance of the bradykinin and bradykinin fragments at different retention times is further evidence for degradation in solution, rather than in the mass spectrometer. An interesting feature appearing in the LC/MALDI-MS topographic plots of Figures 4 and 5 is the decrease in lowmass ion signal while an analyte is eluting from the column. The reduction of low-mass ion signal is particularly evident at 13-, 19-, and 30-min retention time at flight times between 3 and 10 ps. This effect has been observed previously25and can be attributed to one or more processes. It is possible, for example, that the number of positive charge carriers produced by aerosol MALDI is constant and that the production of ions with longer flight times requires a decrease in ions with shorter flight times. A second possibility is that the presence of analyte in the aerosol somehow lowers the efficiency of the ionization process and hence the total ion signal. This second possibility
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Analytical Chemistry, Vol. 66, No. 10, May 15, 1994
is plausible in light of results pertaining to solvent evaporation and peptide and protein inclusion in MALDI matrices.27 Further investigation of this phenomenon is warranted. CONCLUSIONS We have demonstrated LC/MALDI-MS with a separation of nanomole quantities of bradykinin, gramicidin S, and myoglobin. Several interesting features in the HPLC separation were identified. HPLC peak splittings were observed and attributed to solution complexes of bradykinin-myoglobin and gramicidin S-gramicidin S. The Arg-Pro and Pro-GlyPhe-Ser-Pro-Phe-Arg fragments of bradykinin were observed from what appears to be chemical degradation on or before the HPLC column. A decrease in low-mass ion signal concurrent with high-mass analyte ion formation has been noted and may ultimately yield insight into the ionization process. We are currently investigating the possibility of using aerosol MALDI as a probe of noncovalent interactions of proteins in solution. Instrumental modificationsto improvedata acquisition rate and detection limit are being implemented. Several of these modifications have been discussed previously in detail.22 Modifications that will improve the detection limit include an increased laser duty cycle, more efficient analyte transport to the aerosol-laser interaction volume, a larger laser-aerosol interaction volume, more efficient aerosol drying, and an increase in ion collection efficiency. The increased laser duty cycle will also improve the data acquisition rate. For example, a commercially available 500-Hz excimer laser could poten(27) Murray, K. K.; Russell, D. H. In Laser Ablatiort Mechanisms and Applicarions-ZI; Miller, J. C., Geohegan, D. B., Eds.;AIP Conference Proceedings 288, American Institute of Physics: New York, 1994;pp 459464. (28)Chait, B. T.;Kent, S.B. H. Science 1992,257, 1885-1893. (29)Giddings, J. C. Anal. Chem. 1984,56, 1258A-1270A. (30) Bushey, M.M.;Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (31) Bushey, M. M.; Jorgenson, J. W. A n d . Chem. 1990,62,978-984. (32)Lemmo, A. V.;Jorgenson, J. W. Anal. Chem. 1993,65,1576-1581.
tially reproduce the data shown in Figure 4 in less than 40 s. We are also developing an interface between aerosol MALDI and a Fourier transform mass spectrometer. The advantages of aerosol MALDI for LC/MS are (1) rapid data acquisition with TOF mass analysis, (2) acquisition of an entire mass spectrum on every laser shot, and (3) the ability to detect large biomolecules. Additionally, MALDI has a high tolerance for impurities such as salts and buffers used in LC separations.2s The results reported in this article demonstrate the power of LC/MALDI-MS as a 2D separation method for proteins. The analytical utility of LC/MALDIMS compares favorably with liquid-based 2D separation methods.29 Recent examples involving LC, CE, and size exclusion chromatography (SEC) include LC/LC,30 LC/ CE,)' and SEC/CE.32 TOF-MS combines sensitive LC detection with a second separation dimension, eliminating the need for a second liquid separation. The mass spectrometer permits detection of coeluting analytes, in this case bradykinin and myoglobin coeluting as a result of the formation of a noncovalent complex. ACKNOWLEDGMENT Funding for this research was provided by the Texas Advanced Technology Program and by the Division of Chemical Sciences, Office of Basic Energy Sciences, US. Department of Energy under Grant DE-FG05-85ER- 13434. The Nd:YAG laser and additional equipment were purchased with funds provided by the National Science Foundation under Grant CHE-9223629. Recelved for review December 17, 1993.' 1994
Accepted March 3,
Abstract published in Aduance ACS Absrracrs, April 1, 1994.
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