Anal. Chem. 1005, 65, 493-495
40s
CORRESPONDENCE
Continuous-Flow Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Liang Li,’ Alan P. L. Wang, and Larry D. Coulson Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 that the flow probe interface does not introduce significant peak W i g and memory effect. We envision that further development of the technique described here w i l l make it poesible to combine various solution separation methods such as liquid chromatography (LC) and capillary electrophoresis (CE) with MALD for on-line detection of high molecular weight biochemicals.
Matrix-assisted laser desorption (MALD) has recently become an increasinglyimportant technique for ion generation in mass spectrometry.l+ The technique involves mixing a proper matrix with samples such as peptides and proteins on a substrate, followed by laser desorption. In a time-of-flight mass spectrometer, molecular ions can be observed with little fragmentation even for proteins as large as 300000. At present, only one other technique, electroepray,” in which multiple-charged species are generated from a solution, can be used to ionize such large molecules. It is recognized that due to the difference in the process of ion formation, both electrospray and MALD will be rather complementary with inherent advantages and limitations.8 For complex mixture analysis it is often necessary to perform separation prior to the sample introduction into a mass spectrometer. This work was motivated by the desire to combine various solution separation methods with MALD for biochemical analysis. Inspired by the success of the continuous-flow probe for interfacing solution separation methods with fast atom bombardment mass spectrometry (CF-FAB),”13 we have developed a continuous-flow MALD (CF-MALD) system with the use of a liquid matrix, namely, 3-nitrobenzyl alcohol (3-NBA). Thismethod uses a flow probe to deliver sample and matrix through a capillary tube and onto a stainless steel frit, upon which laser desorption/ ionization is carried out. We report here some preliminary results on CF-MALD. The significance of this work is that we demonstrate that solutions of peptides and proteins can be continuously introduced into a time-of-flight mass spectrometer via the flow probe and ionized by MALD. By examining the results from flow injection analysis with CF-MALD, we also show
EXPERIMENTAL SECTION The experimental setup consists of a simple, custom-made linear time-of-flight mass spectrometer (TOFMS).14 The ionization region of the TOFMS consists of three accelerationplates, namely, the repeller, the extraction grid, and the ground plate, with 1-cm separation (see Figure 1). At 5 cm above the ground plate, two parallel stainlesssteel plates (3 in. long and 1in. wide) are used to deflect the low-mass ions in order to avoid the saturation of the ion detector. A pulse of 500 V with 2-pa width and 600-ns rise and fall times is applied to the deflector. This pulse is delayed by 1.4 ps with respect to the desorption h r pulse. The flow probe is placed in between the repeller and extractiongrid, and it is grounded. With this configuration,high voltages up to 15 kV can be applied to the acceleration plates without electric breakdown. In general, 15 kV is applied to the repeller and 12 kV to the extraction grid for ion acceleration. Ions are detected by using a multichannel plate detector (Galleo Electro-Optics Corp., Sturbridge, MA). The transient signal, are recorded by the LeCroy 9400A digital oscilloscope. Data are then transferred to a PC for processing with the use of a data system developed in house. The data system is capable of transferring and storing transients up to 20K data points at a repetition rate of greater than 10 Hz from the oscilloscopeto the PC via GPIB. The design concept of the flow probe is similar to the frit-type probe used in CF-FAFLIOJS A silica capillary tube (75-pm i.d., 363-pm o.d., 1m long) (PolymicroTechnologies, Phoenix, AZ) is inserted to a 1.27-cm-0.d.and 0.635-cm-i.d. stainlesssteel tube andextendstotheprobetip (=Figure 1). Forelectrichulation, the top section of the probe (-7.62 cm long) is made of Vespel. The probe tip (5-mmo.d., 1-mmi.d., and -5 mm long) also made of Vespel is then screwed onto this insulator. A septum is placed in between the probe tip and the insulator for vacuum sealing. The capillary tube punctures through this septum and is placed about 1mm away from the surface of the tip. A 1.65-mm hole is drilled in the center of the tip face to a depth of 1 mm for housing the stainlesssteel frit (1.59-mm 0.d. and 0.794 mm thick) (Chromatographic Specialities Ltd., Brockville, ON). The frit is pushed into the hole to make a direct contact with the end of the capillary tube. A fiiter paper is wrapped around the probe tip to absorb the excess liquid. The flow probe is inserted into the TOFMS via a custom-made solid probe lock. All chemicals are purchased from Sigma Chemical Co., St. Louis, MO, and used without further purification. The catalog numbers (1992) are M1882 for myoglobin, BO125 for bacitracin,
-
(1) Karas, M.; Bachmann, D.; Bahr,U.; Hillenkamp, F. Int. J. Mass Spectrom. Zon Processes 1987, 78, 53. (2) Tanaka, K.;Waki, H.; Ido, Y.; Akita, S.;Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988,2, 151. (3) Beavis, R.C.; Chait, B.T. Rapid Commun. Mas8 Spectrom. 1989, 3,233; 1989,3,432. Beavis,R. C.; Chait, B. T. Anal. Chem. 1990,62,1836. (4) Nelson, R. W.; Rainbow, M. J.; Lohr, D. E.; Williams, P. Science 1989,246,1585. (6)Spengler, B.;Cotter, R. J. Anal. Chem. 1990, 62, 793. (6) Hdenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. l991,63,1193A and references cited therein. (7) Fenn, J. B.;Mann,M.;Meng, C. K.; Wong, S. F.; Whitehouse, C. M.Science, 1989, 246, 64. (8)Karas, M.; Bahr,U.; Giebmann, U. Mass Spectrom. Reu. 1991,10, 336. (9) Caprioli, R. M. Anal. Chem. l990,62,477A and references cited therein. (10) Ito,Y.; Takeuchi, T.; Ishi, D.; Goto, M. J. Chromatogr. 1985,346, 161. (11) Caprioli, R. M.; Moore, W. T.; Martin, M.; DaGue, B.B.; Wilson, K.; Moring, S. J. Chromatogr. 1989,480, 247. (12) Moeeley, M. A.; Deterding, L. J.; de Wit, J. S. M.; Tomer, K. B.; Kennedy, R. T.;Bragg, N.; Jorgenson, J. W.Ana1. Chem. 1989,61,1577. (13) Suter, M.J.F.; Caprioli, R. M. J. Am. SOC.Mass Spectrom. 1992, 3, 198. 0003-2700/83/0365-0483$04.00/0
(14) Wang, A. P.L.; Li, L. Anal. Chem. 1992,64, 769. (16) Zhang, J.Y.;Nagra,D. S.;Li,L.MolecularCoolingandSupereonic Jet Formation in Laser Desorption. Submittad for publication.
Ca
1883 American Chemical Soclety
m4
ANALYTICAL CHEMISTRY, VOL. 65. NO. 4, FEBRUARY 15, 1993
TO flight Tube
c
I n
-...............-
Extraction Grid
Laser Beam I
Capillary Tube
I
septum
Stainless S t e e l Tube
F i l t e r Paper
Flgum 1. schematic of the continuousflow matrlx-asslsted laser deemption tlim-of-fllght ma88 spectrometer. Drawing is not to scab. The dimensions of the major components are given in me text.
and I5500 for insulin. Since the purities of these samples are un!inown,thesampleconcentrationsshownherearetheestimated value calculated by assuming they were pure. The peptides and proteinsare dinaolved in 0.1 % trifluoroaceticacid (TFA)aqueous solution. A mixture of this solution, methanol, ethylene glycol, and 3-NBA (1:kkl) is continuously introduced onto the tip of theflowprobeataflowrateof 1-5rWminbyusingamicroay~inge pump (Orion Research Inc., Boaton. MA). Alternatively, a 60nL sample injector (Valco Instruments Co., Houston, TX) is placed in between the pump and the flow prohe to perform flow injection analysis. In this case, the carrier solvent is made of 0.1% TFA, methanol, ethylene glycol, and 3-NBA (kk1:l) and the sample solution containing 3-NBA is injected into the flow. To maintain a stable flow,a point heater made of a Nichrome 60 heating coil (Pelican Wire Co., Naples, FL) is placed perpendicular to both the prohe and the flight path to supply gentle heat to the liquid. The distance between the heater and the probe tip is ahout 2 in. The temperature near the point heater is ahout 100 "C. The temperature of the prohe is not monitored in the present design. With a 6 i n . diffusion pump for pumping the ionization region and a 4-in. pump for the 1-m flight tube, the pressure near the ion detector is s h u t 6 X lo+ Torr when the liquid flow rate of up to 5 rllmin is used. No liquid nitrogen traps are used. A NdYAG laaer (Spectra-Physics,Mountain View,CA)is used for performing MALD. The ionization wavelength is 266 nm, and the repetition rate is set at 10Hz. The laser beam is focused hyusingaconvexlens(300-mmfocallength)tna0.5-mm-diameter spot. The laser power densityused varies from 1Wto 107Wlcm2.
RESULTS AND DISCUSSION Figure 2 shows the comparison between the maea spectra of myoglobin obtained by using the solid probe and the flow probe. The mass spectra appear to be similar in both static and dynamic modes. Two findings are worth noting. First, CF-MALD can provide a stable signal for hours whereaa the ionsignallastsseveralminutesinthestaticmode. Theshorter lifetime of the signal observed in the static mode is mainly due to the rapid evaporation of the liquid matrix a t the elevated temperature used. If the static mode is operated a t the room temperature with a lower desorption rate (0.5 mm in diameter) while maintaining a constant laser power density, we do not observe any signal enhancement from the desorption process. The reason for this is presently unknown. It should also be noted that, by comparing the
MALD mass spectra of several peptides and proteins reported in the literature'+ with those obtained with our system operating in the static mode (e.g., Figure 2(A)), we find that the detection sensitivity of our mass spectrometer is much lower. Clearly, more work is needed to optimize the present mass spectrometric system as well as the operational conditions to increase its overall detection sensitivity for MALD. The major limitation of the present experimental configuration for CF-MALD is low mass resolution. The resolution is generally about 10 calculated from t/(2At), where t is the ion flight time and At is the peak width at fwhm. However, we observe at least a 10-fold increase in resolution for small molecules (molecular weight < 1OOO) with MALD by using our existing reflectron TOFMS.le1* We plan to add a reflector, capable of handling higher reflecting voltages, to the linear TOFMS used here to improve the mass resolution to study large biochemicals.
ACKNOWLEDGMENT
This work was supported by the Natural Sciences and Engineering Research Council of Canada. We thank Dr. N. J. Dovichi for the use of the microinjector and Dr. P. Kebarle for the use of the syringe pump. This work was presented in part at the 40th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, May 31-June 5, 1992, Washington, DC. (16)Li,L.;Hogg,A.M.;Wang,A.P.L.,Zhang,J.Y.;Nagra,D.S.Anol.
Chem. 1991,63,974. (17) Nagra, D.S.; Zhang, J. Y.;Li,L. Anal. Chem. 1991, 63, 2188. (18) Wang, A. P. L.;Li,L. Appl. Spectrosc. 1991,45,969.
Received for review August 12, 1992. Accepted October 13, 1992.
