Anal. Chem. 1995, 67, 1998-2003
Mass Resolution improvement by incorporation of Pulsed ion Extraction in a Matrix-Assisted Laser Desorption/ionization Linear Time-of-Flight Mass Spectrometer Robert S. Brown* and John J. Lennon Department of Chemistty and Biochemistry, Utah State University, Logan, Utah 84322-0300
A linear time-of-flightmass spectrometerhas been modified to incorporate pulsed ion extraction of matrix-assisted laser desorptiodionization (MALDI) generated ions. A unique aspect of the experiments presented is the combination of pulsed extraction with very high source potentials (up to 25 kv) which allows improved mass resolution while maintaining excellent sensitivity for the large mlz ions generated by the MALDI technique. Mass resolution in excess of 1000 (fwhm)is demonstratedfor cytochrome c (12 36 1.1 Da) with the pulsed ion extraction linear time-of-flightmass spectrometer described. The influence on obtainable mass resolution of experimental variables such as delay time between laser ionization and ion extraction, amplitude of the pulsed voltage employed, and the source bias voltage are presented. It is shown that, for any given source potential, the optimum pulsed extraction voltage is a linear function of the mass of the analyte. This is consistent with the observation that the initial ion velocity distributionfor WI-generated ions is independent of mass. Matrix-assisted laser desorption/ionization (MALDI) has developed into one of the more important mass spectrometric methods for the analysis of large biologically relevant molecules.1-5 While this ionization technique has been coupled to a variety of mass analyzers including scanning sector instruments equipped with array detectors6 and magnetic ion traps,7J by far the most popular mass analyzers for this technique have been those which utilize time-of-flight WF) mass analysis. This is due to both their relatively simple and inexpensive design and their excellent sensitivity and high-mass capability. Both linear and reflectronbased TOF mass spectrometers (MS) are widely employed. Linear systems combine excellent sensitivity and simple operation (1) Karas, M.; Ingendoh, A; Bahr, U.; Hillenkamp, F. Biomed. Enuiron. Mass Spectrom. 1989, 18, 841-843. (2)Karas, M.; Bahr, U.; Ingendoh, A; Nordhoff, E.; Stahl, B.; Strupat, IC; Hillenkamp, F.Anal. Chim. Acta 1990,241, 175-185. (3) Beavis, R. C.; Chait, B. T. Rapid Commun. Muss Spectrom. 1989,3,432-
435. (4) Beavis
R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990,87, 6873-
6877.
(5) Stahl, B.; Steup, M.; Karas, M.;Hillenkamp. F. Anal. Chem. 1991,63,14631466.
(6) Hill, J. A; Annan, R. S.; Biemann, IC Rapid Commun. Mass Spectrom. 1991,
5,395-399. (7)Castoro, J. A,; Koster C.; Wilkins, C. L. Rapid Commun. Mass Spectrom. 1992,6, 239-241. (8) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993,65,2621-2627. 1998 Analytical Chemistry, Vol. 67, No. 73,July 7, 7995
with demonstrated upper mass limits in excess of 200 000 Da.9 Linear systems also provide adequate mass resolution ( m / A m FZ 500 or less depending on the ion m/z) for many applications.TOFMS, which incorporates an ion mirrorL0to correct for the initial ion velocity spread of W I - g e n e r a t e d ions, can produce a mass resolution of several thousand for ions up to several thousandll m/z units. An ion mirror based system’s mass resolution becomes limited by the increased isotopic distribution of larger m/z ions and, with larger m/z MALDI-generated ions, the high degree of metastable fragmentation which occurs after ion acceleration and prior to the ion mirror. The loss in sensitivity often encountered with a reflecting TOF-MS relative to a linear TOF-MS must also be considered when deciding upon which mode to employ. Current MALDI TOF-MS (both linear and reflector) designs rely on the short laser pulse (typically 1-5 ns, depending on the laser) to produce discrete ion packets in the ion source, which are then continuously extracted from the ion source by the application of a large static electric potential (25-30 kv). The resulting high ion velocity provides for good ion detection efficiency and, especially in the case of the h e a r design, minimizes the effect on mass resolution of the initial velocity distribution. Utilization of dynamic extraction fields has historically been employed for improved mass resolution in TOF-MS.12-14 Pulsed ion extraction methods have also been utilized15-17with laser desorption TOF-MS to compensate for the initial velocity and temporal and spatial distributions of the ions produced. While useful for smaller m/z ions, this approach has generally been abandoned for large m/z MALDI-generated ions in favor of the better mass resolution and sensitivity afforded by continuous ion extraction at high accelerating potentials. A TOF-MS design that can combine both the advantages of pulsed ion extraction and high ion source accelerating potentials offers significant advantages over conventional designs. In this (9)W a s , M.; Bahr, U.;Ingendoh, A.; Hillenkamp, F. Angew. Chem., Znt. Ed. Engl. 1989,28, 760-761. (10)Mamyrin, B. A; Karatajev, V. J.; Shmikk, D. V.; Zagulin, V. A Sou. Phys. JETP 1973,37,45-48. (11) Spengler, B.; Kaufinann, R Analusis 1992,20, 91-101. (12) Wiley, W. C.; McLaren, I. H. Rev. Sei. Znstmm. 1955,26,1150-1157. (13) Browder, J. A.; Miller, R L.; Thomas, W. A.; Sanzone, G. Znt. J. Mass Spectrom. Zon Phys. 1981,37,99-108. (14)Kinsel, G. R.; Grundwuenner. J. M.; Grotemeyer. J. J Am. SOC.Muss Spectrom. 1993,4, 2-10, (15) Spengler, B.; Pan,Y.; Cotter, R J.; Kan, L.3. Rapid Commun. Mass Spectrom. 1990,4,99-102. (16)Wang, B. H.; Dreisewerd, IC; Bahr, U.; Karas, M.; Hillenkamp, F. J. Am. SOC.Muss Spectrom. 1993,4, 393-398. (17) Colby, S. M.; King, T.B.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1994, 8, 869-875. 0003-2700/95/0367-1998$9.00/0 0 1995 American Chemical Society
Nitrogen Laser 337.1 nm and 600 ps (fwhm)
n +300 V
Sample Probe Tip XY Deflector 340 ns to 4 ps
GI Voltage
-100
W-W
Dual MCP Detector
v
n
pulse voltage (0-3 kV max)
W-W
bias voltage (24 kV m a )
4 0 ps
G2
Voltage
= Bias Voltage
Figure 1. Schematic representation of linear TOF-MS used for pulsed ion extraction studies.
paper, the technical design and performance of a high-voltage (0-3 kv) pulsed extraction system capable of being biased at up to 30 kV coupled to an otherwise conventional linear TOF-MS is described. The effect of various fundamental experimental variables (pulse voltage, bias voltage, delay time, analyte m/.)on the obtainable mass resolution of MALDI-generated ions has been studied, and optimal experimental parameters have been determined. Direct comparison of the mass spectra obtained with the same linear TOF-MS operating in conventionalcontinuous ion and optimized pulsed ion extraction (both at equivalent total ion energies) is presented. EXPERIMENTAL SECTION
Instrumentation. The general design of the linear TOF-MS employed in these studies has been described previously.18 Modifications to the system to facilitate pulsed ion extraction include the use of a dual-microchannelplate detector (R. M. Jordan Co., Grass Valley, CA) for improved time response. The singlestage wire ion guide previously describedIg has been modified by the addition of a short (30 cm) second stage prior to the detector to facilitate pulsed ion deflection and prevent detector saturation. Total flight path is now -2.3 m. The transient digitizer’ssampling rate has been increased to 200 MS/s (LeCroy Model 8828A, Chestnut Ridge, NY). The current ion source design utilized for both continuous and pulsed ion extraction is shown in Figure 1. It is a standard threegrid design with spacings of 4 mm between the pulsed grids and 16 mm between the second and third (grounded) grids. Each grid utilizes 90%transmissive mesh to ensure uniform electrostatic extraction fields. As in the previous design, sample is applied to a spring-loadedstainless steel probe tip which mates flush with the first solid ion grid. Limitations in the current electrical isolation of the ion source limits operation to a maximum source bias potential of 24 kV without intermittent arcing. The remainder of the system is capable of operation up to the 30-kV design limit of the pulsed ion extraction electronics (see below). Typical background (18) Brown, R S.;Gilfrich, N. L. Rapid Commun. Mass Spectrom. 1992,6,697701. (19) Brown, R. S.; Gilfrich, N.L. Anal. Chim. Acta 1991, 248, 541-552.
pressures in the TOF-MS ion source during operation are 2 x Torr. A pulsed nitrogen laser (Model PL2300, €TILasers, Princeton, NJ) producing 600-ps (fwhm) laser pulses at 337.1 nm was used for all of the studies presented. Pulsed ion extraction was accomplished by the use of a variable-amplitude (0-3 kV) pulser board (Model GRX 3.0K, Directed Energy, Inc., Fort Collins, CO) biased at high voltage (f30 kV maximum). A 30-kV isolation transformer provides floated 110 VAC power for both the pulser and the auxiliary 0-3 kV high-voltage power supply (Series 602C, Bertan High Voltage, Hicksville, NY) . All components (except the 0-30-kV bias power supply) are in an electrically isolated enclosure. The bias voltage from the variable 30-kV power supply serves as the ground reference for all components. The 0-3-kV pulse voltage for positive ion extraction is connected with -2 ft of RG59 cable via a high-voltage vacuum feedthrough to the first source grid, and the variable bias voltage is connected to the second grid. Pulse rise times with this arrangement and the GRX 3.OK pulser (unbiased) were measured to be -20 ns. In the positive ion mode experiments described here, when the pulser is triggered after a variable delay period relative to ion formation, the increased positive potential on the first source plate repels the ions out of the first source region. They are then accelerated to their final velocity through the second grid region, which is held at a constant potential difference (equal to the bias voltage employed). Although not demonstrated here, negative ion spectra can be generated by the use of a negative polarity biasing supply and interchange of the potentials applied to the first two source grids. The pulse voltage employed can be varied (0-3 kv) by adjusting an electrically isolated potentiometer. The pulser itself is triggered via a fiber-optic cable and a simple circuit which provides for control of the pulse delay after ion formation and the pulse duration. The pulse duration employed for all experiments was 10 ,us,which ensured all of the ions formed had been accelerated out of the ion source. Pulse delays between 320 and 1060 ns were utilized. All events (digitizer and pulser) were triggered from the response of a dc biased silicon pin diode (
