Anal. Chem. 1997, 69, 4390-4398
Laser Photodissociation of Insulin Ions Generated by Matrix-Assisted Laser Desorption Jan Preisler† and Edward S. Yeung*
Ames LaboratorysUSDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
A novel method for studying the ionization step of matrixassisted laser desorption/ionization (MALDI) is demonstrated. A 193-nm pulse from an ArF excimer laser is used to photodissociate a portion of a plume of insulin ions generated by MALDI. Laser photodissociation (LPD) creates a “hole”, i.e., a negative spike in the insulin peak in the time-of-flight (TOF) mass spectrum. The position of the hole in the mass spectrum provides useful measurements of the characteristics (position, time, and velocity) of insulin ions shortly after their creation. Although the performance of the method can be further improved, the data obtained could be used to refine our current understanding of MALDI and to improve the resolution of MALDI-TOFMS. Matrix-assisted laser desorption/ionization1 (MALDI) has become an important ionization method for protein analysis by mass spectrometry (MS). The method developed quickly during the first decade of its existence. The applications of MALDI were extended to nucleic acids, saccharides, and synthetic polymers.2-4 Although the pulsed character of MALDI makes it an ideal ionization method for time-of-flight (TOF) mass spectrometers, the technique is also compatible with other instruments. Particularly, utilization of FTICR-MS has led to a large enhancement of resolution and accuracy of mass determination.5,6 The combination with TOFMS will probably remain the most attractive, especially since time-delay focusing has been shown to improve the performance.7-10 The major disadvantage of TOFMS is its limited mass resolution. Ideally, ions with equal velocities are formed instantaneously on an equipotential plane in the extraction electric field. The time of flight of such ions would only be dependent on their m/z ratio, and the instrument would show resolution limited only by the † Present address: Department of Chemistry, Barnett Institute, Northeastern University, Boston, MA 02115. (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (2) Hillenkamp, F.; Karas, M.; Beavis, R.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1203A. (3) Karas, M.; Bahr, U.; Giessmann, U. Mass Spectrom. Rev. 1991, 10, 335357. (4) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R. (5) McIver, R. T., Jr.; Hunter, R. L. Int. J. Mass Spectrom. Ion Processes 1994, 132, L1-L7. (6) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (7) Colby, S. M.; King, T. B.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1994, 8, 865-868. (8) Whittal, R. M.; Li, L. Anal. Chem. 1995, 67, 1950-1954. (9) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003. (10) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946.
4390 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
time response of the detector. However, there are numerous factors which reduce the resolution of real TOFMS. These factors include inhomogeneous electrical field, space-charge effects, insufficient speed of the ion detector and signal recorder, finite time of ion formation, postsource decay of ions, nonuniform velocities (energies), and spatial spread of ions. Proper construction and alignment of the ion optics (plates and grids) can make the electric field homogeneous. Space-charge effects can be minimized by the formation of a limited number of ions, e.g., by using low laser powers. The time response of commonly used detectors for TOFMS, such as MCP, is already at the subnanosecond range. Fast 1-GHz A/D converters or high-frequency pulse counting techniques are already available. Thus, if the postsource ion decay11 is excluded, the dominating factors limiting the resolution of analytical TOFMS are the spatial, velocity (energy), and temporal distribution of the ions generated during the MALDI process. Despite the many possible mechanisms of ion formation,12-14 only a fraction of the analyte molecules desorbed by MALD is ionized. An increase in ionization efficiency cannot be achieved by simply increasing the desorption power density because that also promotes fragmentation. Furthermore, excess production of ionized fragments of matrix may saturate the detector and complicate the mass spectra. In fact, the best results are always achieved with power densities only slightly above the threshold.15 Another problem in the ionization step is that some of the desorbed species might be ionized more efficiently than others. Deeper knowledge of the dynamics involved in the desorbed plume would be useful for the possible incorporation of postionization, so that ionization could be decoupled from the desorption process and optimized independently to improve the overall performance. Currently, ion formation is assumed to take place in the condensed phase or in a dense plume formed near the sample surface immediately following the desorption laser pulse.12 A large velocity distribution is considered to be the major source of the peak broadening in MALDI-TOFMS. Beavis and Chait found large velocity distribution with an offset velocity of approximately 750 m/s for the ions of three proteins and about 1100 m/s for matrix ions.16 Pan and Cotter reported a similar velocity for matrix (11) Spengler, B.; Kirsch, D.; Kaufman, R. Rapid Commun. Mass Spectrom. 1991, 5, 198-202. (12) Spengler, B.; Kaufmann, R. Analusis 1992, 20, 91-96. (13) Yeung, E. S. ChemtractssAnal., Phys. Inorg. Chem. 1990, 2, 103-117. (14) Wang, B. H.; Dreisewerd, K.; Bahr, U.; Karas, M.; Hillenkamp, F. J. Am. Soc. Mass Spectrom. 1993, 4, 393-398. (15) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 233237. S0003-2700(97)00248-5 CCC: $14.00
© 1997 American Chemical Society
molecules and different velocities for two proteins (of similar mass) of 500 and 1000 m/s. The velocities estimated by other workers are close to the velocities above.17-20 Some of the findings, such as that determined from the energy deficit,20 suggest that the real situation is more complex and other sources of broadening need also be considered. Several methods have been proposed to reduce the effects of the initial velocity distribution in MALDI-TOFMS. Wiley and McLaren’s dual-grid design21 allows independent adjustment of the magnitude of the extraction field in the ion source region at a constant overall acceleration potential. Under certain conditions, this enables one to reach a compromise between different types of ion distributions. In MALDI, however, the best resolution is achieved at the highest electrical field in the extraction region.15 The same authors also introduced the concept of time-delay extraction, which consists of insertion of a time delay between ion formation and the application of the extracting pulse.21 During the delay period, the ions travel toward or away from the detector. After the voltage on the repeller is increased, the ions which moved fastest toward the detector are closer to it and receive a smaller amount of acceleration, and vice versa. Thus, the spatial distribution at the time of the electrical pulse corrects for the initial velocity distribution. This approach works well if the original spatial distribution is negligible. Time-delay extraction was demonstrated to increase the mass resolution of proteins and DNA.7-10 Special consideration of the correlation between ion position and velocity is given in ref 7. The authors emphasized the fundamental difference between the laser desorption/ionization method and conventional gas-phase ionization methods. The spatial and velocity distributions are independent in gas-phase MS, while they are correlated in the LDI-MS. An alternative way to increase the resolution is to eliminate the velocity distribution and correct for the spatial distribution.22 Other forms of time-dependent acceleration fields can be employed.23 Another popular way to correct for the initial velocity distribution is the use of the reflectron TOFMS.24,25 In this case, an ion mirror is arranged such that the ions with larger velocities have to travel greater distances than the slower ones. Disadvantages of the reflectron TOFMS are its higher complexity and lower ion transmission, although a gridless instrument26 can be used. Previously, we have used several instrumental approaches to study laser-generated plumes. A fast absorbance imaging system was used for imaging of atoms,27 diatomics,28 and larger molecules.29-31 Absorbance and fluorescence monitoring were successfully applied to study plumes of proteins and DNA (16) Beavis, R. C.; Chait, B. T. Chem. Phys. Lett. 1991, 181, 479-484. (17) Pan, Y.; Cotter, R. J. Org. Mass Spectrom. 1992, 27, 3-8. (18) Spengler, B.; Cotter, R. J. Anal. Chem. 1990, 62, 793-796. (19) Ens, W.; Mao, F.; Mayer F.; Standing, K. G. Rapid Commun. Mass Spectrom. 1991, 5, 117-123. (20) Zhou, J.; Ens, W.; Standing, K. G.; Verentchikov, A. Rapid Commun. Mass Spectrom. 1992, 6, 671-678. (21) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150-1157. (22) Opsal, R. B.; Owens, K. G.; Reilly, J. P. Anal. Chem. 1985, 57, 1884-1889. (23) Kinsel, G.; Grundwuerner, J. M.; Grotemeyer, J. J. Am. Soc. Mass Spectrom. 1993, 4, 2-10. (24) Mamyrin, B. A.; Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A. Sov. Phys.JETP 1973, 37, 45-48. (25) Boesl, R. B.; Neusser, H. J.; Weinkauff, R.; Schlag, E. W. J. Phys. Chem. 1982, 86, 4857-4863. (26) Boesl, U.; Weinkauf, R.; Schlag, E. W. Int. J. Mass Spectrom. Ion Processes 1991, 112, 121-166. (27) Steenhoek, L. E.; Yeung, E. S. Anal. Chem. 1981, 53, 528-532. (28) Huie, C. W.; Yeung, E. S. Spectrochim. Acta 1985, 40B, 1255-1258.
generated by MALD.31,32 Optical probes are able to monitor all absorbing, fluorescing, or scattering species, including neutral molecules, directly at the site of desorption and without interfering interactions with the electrical field. So, they can provide insights into the mechanisms of desorption and ionization separately. However, because of lower sensitivity and limited temporal resolution, optical probes may not accurately reflect the events in mass spectroscopy. The laser power and the size of the desorption spot were larger than those in the case of MALDITOF. Because of the limited rate of imaging, the experiments had to be done at finite gas pressures in order to slow down the development of desorbed plumes. In this work, we use a pulsed 193-nm laser beam for destructive probing of a plume of insulin ions generated by MALDI. Photodissociation of large molecules is much more probable than their photoionization via multiphoton processes.33-35 This is particularly true in the case of 193-nm radiation, which is known to photodissociate peptides very efficiently.36-39 The experimental setup is similar to those for postionization of neutrals generated by laser desorption with one important difference. Instead of increasing the ion signal, we deliberately decrease the ion signal. A pulse of the first, desorption laser generates a normal plume of matrix species and insulin. The insulin ions are accelerated by the electric field in the ion source toward the detector. After an appropriate delay, a second, photodissociation laser is employed to photodecompose a part of the insulin plume. The photodissociated insulin is detected indirectly as a negative spike in the primary insulin peak in the mass spectrum. Knowledge of the time and location of the insulin ions shortly after their creation provides important insights into the ionization mechanism. EXPERIMENTAL SECTION Chemicals and Sample Preparation. 4-Hydroxy-3-methoxycinnamic (ferulic) acid (Aldrich Chemical, Milwaukee, WI) was used as a model matrix on the basis of its proven utility in MALDI. A 0.1 M matrix solution was prepared in a 50:50 ethanol:water mixture. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Bovine insulin (BI) was used as a representative protein for testing MALDI-TOFMS. A 1 mM solution of BI was prepared by dissolving it in 0.1% trifluoroacetic acid. Sample films were prepared on stainless steel tips by simply evaporating the solvent at room temperature and atmospheric pressure. The deposition area of the tip was roughened (sandblasted) in order to improve the coverage of the tip with sample films. The diameter of the deposition area was 6.35 mm. Ten microliters of the sample solution of ferulic acid and insulin (29) Kimbrell, S.; Yeung, E. S. Appl. Spectrosc. 1989, 43, 1248-1251. (30) Heise, T. W.; Yeung, E. S. Anal. Chem. 1992, 64, 2175-2179. (31) Preisler, J.; Yeung, E. S. Appl. Spectrosc. 1995, 49, 1826-1833. (32) Heise, T. W.; Yeung, E. S. Anal. Chem. 1994, 66, 355-361. (33) Zare, R. N.; Levine, R. D. Chem. Phys. Lett. 1987, 136, 593-599. (34) Grotemeyer, J.; Boesl, U.; Walter, K.; Schlag, E. W. Org. Mass Spectrom. 1986, 21, 595-597. (35) Grotemeyer, J.; Boesl, U.; Walter, K.; Schlag, E. W. Org. Mass Spectrom. 1986, 21, 645-653. (36) Bowers, W. D.; Delbert, S.-S.; McIver, R. T., Jr. Anal. Chem. 1986, 58, 969-972. (37) Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III. J. Chem. Soc., Chem. Commun. 1987, 548-550. (38) Hunt, D. F.; Shabanowitz, J.; Yates, J. R., III; Griffin, P. R.; Zhu, N.-Z. Anal. Chim. Acta 1989, 225, 1-10. (39) Williams, E. R.; Furlong, J. J.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1990, 1, 288-294.
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(matrix-to-analyte ratio ) 100) was deposited on the probe tip. The optimum proportion of ethanol in the solutions was found to be approximately 50%. Films consisted of microcrystals uniformly covering the entire deposition area. Deposition of sample solutions with higher (∼1000) matrix-to-analyte ratios resulted in uneven coverage of the probe tip by large, needle-shaped crystals. The film thickness also varied in that case. Mass Spectrometer. The TOFMS was a linear WileyMcLaren type. The probe load mechanism, acceleration optics, flight tube, and detector were purchased from R. M. Jordan Co. (Grass Valley, CA). Both the repeller plate and the extraction grid were powered by two independent power supplies (Spellman, Hauppauge, NY, Model CZE1000R/X2263). This allowed adjustment of the extraction grid potential, Vb, independently of the repeller plate potential, Va, and detection of either positive or negative ions. The (second) acceleration grid was grounded. Transmission of both grids was 90%. The maximum accelerating voltage was 15 kV; Va ) 15.0 kV and Vb ) 0-15 kV were used during the experiments. A 40-mm dual-multichannel plate (MCP) with extended dynamic range offered both excellent ion collection and sensitivity. The potential on the first detector plate, Vd ) -1.96 kV, further increased the kinetic energy of the arriving cations. The transmission of the detector input grid was 82%. The physical dimensions of the instrument were measured with an accuracy of 0.1 mm: ion source region (repeller plate to extraction grid), s ) 12.7 mm; acceleration region (extraction grid to acceleration grid), d ) 15.1 mm; drift region (acceleration grid to detector input grid), D ) 499.7 mm; and detector region (input detector grid to front face of the MCP), S ) 17.7 mm. The thickness of the grids was negligible. The instrument was evacuated using a 15-cm diffusion pump (Varian, Lexington, MA, Model VHS-6) with a maximum pumping speed of 2400 L/s. Oil contamination of the mass spectrometer was prevented by using a cryotrap (Varian, Model 326-6) and an electropneumatic gate valve (MDC, Hayward, CA, Model GV-8000V-ASA-P). The ultimate pressure in the source chamber was 1 × 10-8 Torr; experiments were done at pressures below 1 × 10-7 Torr. A vacuum controller with two thermocouple gauges and one ion gauge was purchased from Granville-Phillips (Boulder, CO, Model 307). Optical Setup. A six-way cross with five interchangeable flanges used as the source chamber of the mass spectrometer allowed implementation of photodissociation. In this case, a flange with a 15-cm S1-UV window served as an entrance for both the desorption and photoionization laser beams. The desorption beam from a 337-nm, 9 mJ/pulse nitrogen laser (Laser Photonics, Orlando, FL, Model UV24) was attenuated by a series of glass plates and directed to the source chamber by a mirror (Figure 1). An aperture and S1-UV lens (f 25 cm) were used to generate the desorption spot (0.2 mm × 2.0 mm) on the probe tip. The angle of incidence of the desorption beam (defined by the beam and the flight axis) was 60°. The pulse energy of the desorption laser was determined with a pyroelectric energy probe (Molectron, Campbell, CA, Model J3-09). The power density in the desorption spot was 2.0 MW/cm2, approximately 20% above the desorption threshold. The desorption laser was focused on one side of the circular probe tip so that a rotation of the probe tip exposed fresh film to the desorption laser and a single probe tip could be used about 20-30 times. 4392 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
Figure 1. Schematic of the experimental arrangement for MALDILPD-TOFMS.
A 193-nm, 40-mJ ArF excimer laser (Lumonics Inc., Kanata, ON, Canada, Model HyperEx-460-HR-A) was used for photodissociation of desorbed plumes. The rectangular beam of the excimer laser offered better focus in the vertical dimension. The combination of a mirror and a quartz plate was used to rotate the laser beam 90° and direct it into the source chamber. This setup enabled better focusing in the direction of flight axis and reduced somewhat the intensity of the laser. A S1-UV lens (f 30 cm) was used to focus the excimer beam in the ion source region several millimeters above the sample probe. A 1-mm-wide slit was inserted behind the lens to decrease the spot width and to further reduce the photodissociation power density. The beam path was parallel to the repeller and the grids; the beam entered the source chamber through the large window and left it through a smaller Suprasil window positioned off-axis on the flange opposite the large window. The average energy of the desorption laser was determined with an energy probe (Scientech, Boulder, CO, Model MC 2501). The power density in the photodissociation spot was on the order of MW/cm2. To measure the size of the photodissociation spot and its distance from the sample film, a piece of thermosensitive paper instead of the probe tip was inserted in the sample holder into the source chamber, and several hundreds of laser shots were applied. The dimensions of the photodissociation focus of 0.1 mm × 3.0 mm were estimated from the spot burnt on the paper. Thus, a relatively narrow slice of the desorbed plume could be irradiated. The spot-to-spot distance (the distance between the desorption spot and the photodissociation spot) was calculated by subtracting the probe tip height (9.5 mm) from the position of the spot burnt on the paper. Experiment Control and Data Acquisition. The timing of the experiment (repetition rate, the desorption, and the photodissociation lasers) was controlled by a four-channel digital delay generator (EG&G PARC, Princeton, NJ, Model 9650). Mass spectra were recorded by the first channel of a 500-MHz digital oscilloscope (LeCroy, Chestnut Ridge, NY, Model 9350AM). The second channel was used for acquisition of the signal from a fast photodiode (Hamamatsu 3399) placed behind the exit window. Half of the photodiode entrance window was covered with a fluorescent tape so it responded to light from the both lasers. Thus, the shot-to-shot delay (the delay between the desorption and the photodissociation laser pulses) could be verified. In practice, no correction was necessary, since the jitter of both lasers was better than (2 ns. The photodiode signal corresponding to the desorption laser was used to trigger data acquisition. The repetition rate was 6 Hz. After an adjustment of the proper shot-
to-shot delay, both lasers were turned on. The first 5-7 shots were omitted, and the mass spectra with an insulin peak were recorded with a temporal resolution of 2 ns. Fifty traces were averaged to improve the signal-to-noise ratio. THEORY In MALDI-TOFMS, an ion with a mass m and a charge number z is formed at a distance x0 from the repeller plate with an initial velocity v0 at time t0 after the desorption pulse. The primary insulin ion is MH+, so we used m ) 5735 au (9.523 × 10-22 kg) in our calculations. The time between the pulse from the desorption laser and the subsequent detection of an ion is conventionally called the ion time of flight, t. This includes the delay between the desorption of a neutral species and its ionization, t0, and the time the ion spent in the source region, t1, in the acceleration region, t2, in the drift tube, t3, and in the detector region, t4. So,
t ) t0 + t1 + t2 + t3 + t4 ) t0 +
2(s - x0) 2d D 2S + + + v0 + v1 v1 + v2 v2 v2 + v3
x
v02 +
v2 )
v3 )
2ez(s - x0)(Va - Vb) ms
(1a)
x
2ezVb m
(1b)
x
2ezVd m
(1c)
v12 +
and
v22 -
v0 ) x0/t0
(1)
where
v1 )
Finally, space-charge effects may also cause some distortion from ideal behavior of ions. The first situation (the creation of the ion above the repeller) can be most readily described. In our previous work,31 we determined the axial velocity of a plume of tetramethylrhodamine isothiocyanate-labeled bovine serum albumin generated by MALDI at low pressures. The velocity was constant over a distance of 0.5-8 mm from the sample film. Bovine serum albumin was desorbed predominantly in its neutral form. This was confirmed by later experiments in the presence of an electrical field, where no change was noted compared to the experiment in a field-free environment. Although our experiments could not confirm linear movement within the first 0.5 mm or record temporal delays in desorption, an assumption of constant velocity for neutrals after desorption and the lack of a delay in desorption may be reasonable descriptions of the real situation. The most abundant form of an analyte ion in MALDI is the protonated neutral. The collision with a matrix molecule with proton transfer should not significantly change the velocity of the analyte. Thus,
e is element charge, v1 is the ion velocity at the extraction grid, v2 is the ion velocity in the drift tube, and v3 is the ion impact velocity on the MCP. Explanation of the other variables can be found in the Experimental Section. Knowledge of t alone cannot be used for calculations of all unknowns, i.e., x0, v0, and t0. Also, varying the extraction field strength via Vb may not yield the right ionization characteristics because ionization may be dependent on the extraction field strength. As mentioned earlier, peak broadening in MALDI is usually attributed to large initial velocity distributions. In this case, the analyte is supposed to be ionized in the sample film or close to it shortly after the desorption pulse, i.e., x0 ) 0 and t0 ) 0 for all ions. This simple model, however, cannot explain some observed phenomena, especially the late arrival of analyte ions at the detector and any energy deficit, because a negative v0 does not have a physical meaning. There may be several reasons for the energy deficit. First, the ion may be formed above the surface, and only a fraction of the total electrical energy in the source region is transferred into the kinetic energy of the ion. Second, the analyte ion loses part of its kinetic energy because of collisions with a plume of neutrals. Third, electrons originating mainly from the ionization of matrix molecules may create a negative shield between the repeller and the analyte ions. Lighter positive ions can cause similar shielding.
(2)
This states that, with the above assumption, an ion created at a position x0 above the sample film and time t0 after the desorption pulse has a higher initial velocity than an ion formed at the same position later. v0 is simply the velocity of the neutral species at the moment of ion formation. This leads to an energy deficit. However, ions do not have to always show energy deficit. An ion may be created directly on the surface after a delay t0. Then there is no relationship between v0 and t0. This case will be discussed later. Creating a hole (depletion zone) in the plume of analyte ions by photodissociation at a distance xp from the repeller plate at time tp will yield additional information about the movement of analyte ions. For this second event,
2(xp - x0)
tp ) t0 +
x
(3)
2ez(xp - x0)(Va - Vb) v0 + ms
v0 +
2
If the assumptions we made are valid, then eqs 1-3 should give unambiguous solutions for x0, v0, and t0, which characterize the movement of the hole in the plume of insulin ions which was or will be decomposed at the photodissociation spot. Data processing consisted of several steps. First, the ion velocity at the photodissociation spot, vp, was calculated. Equation 1 may be modified in order to describe the movement of the hole from its creation to its detection:
t ) tp + t1′ + t2 + t3 + t4
)
2(s - xp) 2d D 2S + + + vp + v1′ v1′ + v2 v2 v2 + v3
(4)
where t1′ is the time the hole spent in the source region, and
v1′ )
x
vp2 +
2ez(s - xp)(Va - Vb) ms
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The calculated velocity, vp, of the hole at the time of its creation can be transformed into kinetic energy and compared with the kinetic energy of a hypothetical analyte ion formed at x0 ) 0 with v0 ) 0:
∆E )
mvp2 zexp(Va - Vb) 2 ms
(5)
If the difference between the two energies, ∆E, is negative, the ion shows an energy deficit. This may be explained, for instance, by ionization occurring above the surface, as discussed earlier. Knowledge of xp, tp, and vp allows us to use simpler equations for the ion source rather than eqs 1 and 4:
t0 ) tp -
2(xp - x0) v0 + vp
(6)
and
v0 )
x
vp2 -
2ex(xp - x0)(Va - Vb) ms
(7)
The ionization characteristics of an ion may be then calculated from eqs 2, 6, and 7. The solution has physical meaning for x0 in the interval from 0 to xp, t0 from 0 to tp, and v0 from 0 to vp. Ions with an energy excess could also be created above the surface from neutrals, provided that the kinetic energy of the neutral at the point of ionization is higher than the energy that would have been acquired by acceleration of an ion from the surface to that point. This model, however, cannot explain the arrival of ions at the photodissociation spot with energy excess after a certain time limit. In such a case, the ion might be formed directly on the surface, i.e., x0 ) 0, with a velocity v0 after a delay t0. Peak broadening would stem from the velocity and temporal distributions only and the parameters v0 and t0 can be calculated from
v0 )
x
vp2 +
2zexp(Va - Vb) ms
(8)
and
t0 ) tp -
2xp (v0 + vp)
(9)
A program was written to solve both equation sets (either eqs 2, 6, and 7, or eqs 8 and 9), and the meaningful solution was selected, since only one solution for a given t and tp can be correct. RESULTS AND DISCUSSION Sample films with a matrix-to-analyte ratio of 1000 were desorbed during our first experiments. These films did not cover the probe tip area uniformly, and their thickness was up to ∼0.5 mm. As a result, a significant dependence of the insulin peak shape and the peak area on the irradiated sample spot was observed. Because preparation of samples with a matrix-to-analyte ratio of 100 provided much more uniform and thinner films, these samples were selected for further study. Although no shift in the positions of the insulin peak and the negative spike was noticed 4394
Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
when studying samples with matrix-to-analyte ratios of 100 and 1000, systematic study of the influence of the matrix-to-analyte ratio was not done because of the poor quality of the samples with a matrix-to-analyte ratio of 1000 and higher. Samples with matrix-to-analyte ratio of 100 gave more stable and reproducible mass spectra. The insulin signal decreased only slowly within the signal averaging period (50 shots). The power density at the photodissociation spot could be roughly adjusted by changing the laser voltage. It was kept as low as possible. Large photodissociation power densities created too many charged fragments, which could increase space-charge effects and lower the electric field strength due to shielding of the repeller. It was also important to use the 1-mm slit behind the focusing lens to reduce the laser power. Problems such as a change in peak profile, especially if peak tailing was noted, were avoided by reducing the laser power. Peptide bonds absorb 193nm radiation strongly, and cleavage of just one of the bonds was sufficient for our purpose. A power density at the photodissociation spot around 1 MW/cm2 was found to be adequate. The power density of the desorption laser was also kept low, just enough to provide a stable insulin signal with a satisfactory signal-to-noise ratio. The reasons were the same as in the previous case, i.e., prevention of space-charge effects and shielding of the repeller plate. The optimal power density at the desorption spot was 2.0 MW/cm2, which was approximately 20% above the threshold level for detection of insulin. An increase of the desorption power density to 2.5 MW/cm2 resulted in a larger signal, but it did not change the arrival time to the detector or the width of the photodissociation hole. Therefore, space-charge effects are not important in these studies. In order to estimate the effect of electronic shielding, the current from the detector was integrated over 50 µs (mass range up to ∼24 000 au for z ) 1). The extraction grid was grounded to maximize the electric field strength in the source region and, hence, the collection efficiency. After correcting for the gain of the detector (∼106) and transmission losses on the three grids (0.66), the estimated charge created in the source region was 1.5 × 10-15 C (∼104 unit charges). The capacitor formed by the repeller and the plate with the extraction grid has the capacitance of 4.0 pF. The decrease in the repeller voltage, Va, after absorption of all electrons from the ionization process would be less than 0.4 mV. Even if the sample film acts as an insulator and concentrates the negative charges on its surface, the local decrease of the electrical field strength should not be significant. Furthermore, the insulin flight time was not altered, even if the desorption power density increased (with an increase in the insulin peak area) to 4.0 MW/cm2. Such conditions created pronounced peak tailing, which was probably due to space-charge effects. So, any energy deficit occurring at low desorption power density must stem either from ionization above the sample film or from collisions of the analyte ions with the plume of matrix molecules. A set of 18 experiments was carried out. The repeller voltage was kept constant, Va ) 15.0 kV. The photodissociation distance, xp, was adjusted to 1.0, 2.6, and 3.8 mm above the surface. Voltages on the extraction grid, Vb, were set to 0.0, 5.0, 10.0, 13.0, 14.0, and 14.5 kV, which corresponded to electric field strengths in the source region of 11 810, 7870, 3930, 1580, 790, and 390 V/cm, respectively. Portions of insulin (positive) MALDI-TOFMS spectra taken under these conditions are shown in Figure 2. Each spectrum is a 50-trace average, with the photodissociation laser
Figure 2. MALDI-TOFMS spectrum of insulin at different extraction field strengths, E ) (15.0 kV - Vb)/1.27 cm. Vb ) 0.0, 5.0, 10.0, 13.0, 14.0, and 14.5 kV (from left to right).
Figure 4. TOFMS spectrum of insulin at Vb ) 0.0 kV and xp ) 2.6 mm. Traces from top to bottom: MALDI, MALDI-LPD at tp ) 0.51 µs, MALDI-LPD at tp ) 0.61 µs, and difference spectra at tp ) 0.51 and 0.61 µs, respectively.
Figure 3. MALDI-LPD-TOFMS spectrum of insulin under the same conditions as in Figure 2. The photodissociation laser was pulsed at the distance xp ) 2.6 mm with delays tp ) 0.54, 0.64, 0.88, 1.30, 1.75, and 2.3 µs (from left to right).
turned off. Both the height and the resolution of the peak dropped with decreasing electric field strength in the extraction region. We were able to distinguish the insulin pseudomolecular ion from the adducts between insulin and the matrix in those traces with higher resolution. Other possible peaks, such as insulin adducts with alkaline ions or the peak of [M - 17]+, are not resolved. The limiting factor of the resolution at high extraction electric field strength is probably the poor quality of the beam profile at the desorption spot. If the photodissociation laser is turned on, we can observe a negative spike in the insulin peak corresponding to the hole formed in the plume of insulin ions (Figure 3). We can even notice spikes in the peaks of the insulin adduct ion in the first three spectra. The resolution of the spikes is better than the resolution of the original peaks. In these experiments, the photodissociation laser was focused at a distance xp ) 2.6 mm above the repeller plate, and the delay of the second laser was adjusted so that it created a spike approximately at the peak maximum (tp ) 0.54, 0.64, 0.88, 1.30, 1.75, and 2.20 µs in the order of decreasing electric field strength). The spike width increased with the width of the primary peak, which means that the decomposed insulin ions had different initial velocities (as a result
of velocity or spatial broadening). The ratio of the spike height to the peak height is less than 100%. There may be several reasons for this, such as the photodissociation efficiency being lower than 100%, up to 30% fluctuation in the energy per pulse of the photodissociation laser, or presence of ions with several m/z values in the plume. In Figure 3, the ratio of the spike height to the peak height drops at low extraction field strength. The plume arriving at the photodissociation laser spot, especially those arriving later due to a low electric field strength, can become wider than the photodissociation laser spot because of radial diffusion. A gradual, shallow spike suggests wide velocity spreads of the decomposed insulin ions as a result of initial velocity and/or spatial spreads at low electric field strength. Conversely, a sharp, deep spike at high electric field strengths indicates either a single dominant initial distribution or several types of initial distributions with mutual correlation. The role of the spatial and the temporal width of the photodissociation pulse also needs consideration. The photodissociation beam is characterized by a roughly rectangular profile with a spatial width of ∼100 µm and a temporal width of 10 ns (fwhm). The finite spatial width is important especially at short xp (relatively large portion of the plume is irradiated). Indeed, a rectangular profile of the spike was observed under these conditions (xp ) 1.0 mm, Vb > 10.0 kV, not shown). The contribution of the finite temporal profile may be significant at high vp when new ions may enter the photodissociation focal point during the laser pulse. The velocity of an insulin ion at xp ) 3.8 mm will be roughly 1.2 × 104 m/s for Vb ) 0.0 kV, x0 ) 0, t0 ) 0, and v0 ) 0-2000 m/s. At this velocity, the insulin ion travels over a path of 120 µm in 10 ns, which is comparable to the spatial width of the photodissociation laser spot. Different parts of the plume may be photodissociated by altering the delay time between the two laser pulses (Figure 4). The upper trace in Figure 4 is the insulin peak from simple MALDI Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
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Table 1. Linear Regression Fitting to Data of t vs tp at Several Values of Vb and xpa xp (mm) Vb (kV)
p
1.0 q
0.0 5.0 10.0 13.0 14.0 14.5
1.629 1.301 1.078 1.024 1.080 1.105
24.22 24.57 25.18 26.13 27.02 28.10
a
r2
p
2.6 q
r2
p
3.8 q
r2
0.998 0.999 0.992 0.996 0.997 0.996
1.654 1.551 1.243 1.139 1.132 1.190
23.92 24.12 24.70 25.48 26.18 26.91
0.999 0.999 0.999 0.999 0.999 0.999
1.513 1.384 1.184 1.027 1.035 1.051
23.80 24.02 24.52 25.31 25.91 26.62
0.997 0.996 0.998 0.999 0.999 0.999
t ) ptp + q; p, slope; q, intercept; r2, correlation coefficient.
at Vb ) 0 kV. At constant xp ) 2.6 mm, the photodissociation laser was fired at tp ) 510 or 610 ns to create a hole in the front or in the tail of the plume (Fig. 4, second or third trace from the top, respectively). Finally, the two lower traces in Figure 4 represent the difference between the top trace and the spiked traces. They depict the net hole for the insulin photodissociated at tp ) 510 and 610 ns. Variations in the baselines of the two lower traces come from slightly different properties of the sample films employed when recording the top three spectra. In spite of these baseline variations, reasonable estimation of the spike position can be obtained. The time of flight of the hole, t, increases linearly with the pulse delay, tp. Table 1 summarizes the results of linear regression fit of 10-20 data points [tp, t] for all 18 experiments. The 18 experiments were carried out at xp ) 1.0, 2.6, or 3.8 mm and Vb ) 0.0, 5.0, 10.0, 13.0, 14.0, or 14.5 kV. Each of the experiments consisted of 10-20 runs. The value of tp was altered with a step of 10-50 ns over such an interval to create a spike in the center portion of the insulin peak. The time of flight of the hole, t, was then determined for each value of tp. The slope p allows comparison between the temporal distribution of the plume at the photodissociation laser spot and that at the detector. The value of p ) 1 would mean pure temporal broadening. The value of p > 1 stems from velocity spread within the plume and reflects initial velocity or spatial distribution. At certain conditions, the slope can be smaller than 1 or may even be negative, pointing to the presence of a large initial spatial distribution. Thus, we may recognize several broadening mechanisms without additional calculations. At the highest extraction field (low Vb), large velocity distributions are responsible for the peak broadening. The slope is high for all three values of xp. The majority of the velocity broadening occurs at the beginning of the source region. The temporal profile of the plume does not broaden significantly later on because of the leveling effect of the potential of the electrical field on the overall velocity. At low strengths of the extraction field (high Vb), the value p is closer to 1, suggesting more significant temporal broadening. In all cases, the largest value p was found for the intermediate value of xp ) 2.6 mm rather than for xp ) 1.0 mm, which indicates the existence of even more complex processes in the generated plumes. It is also possible that electrical shielding and space-charge effects were underestimated in our analysis. The ion velocity at the photodissociation laser spot, vp, can be calculated according to the eqs 4, 4a, and 1b-d. Data calculated from linear regression was used for t and tp instead of the raw data so that vp at three values of xp could be calculated for a given 4396 Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
Figure 5. Calculated instantaneous velocity vp of the depletion hole at the photodissociation laser spot as a function of pulse delay tp. The hole was created at xp ) 1.0, 2.6, and 3.8 mm for Vb ) 0.0, 9; 5.0, 0; 10.0, b; 13.0, O; 14.0, [; and 14.5 kV, 2. Table 2. Linear Regression Fitting to Data of vp vs tp at Several Values of Vb and xpa Vb (kV)
ttop (ms) aexp (109 m/s2) ath (109 m/s2) bexp (m/s) r2
0.0
5.0
10.0
13.0
14.0
14.5
24.79 19.78 19.84 -465 0.995
25.10 13.23 13.22 -190 0.998
25.79 6.80 6.61 -223 0.999
26.79 2.66 2.64 159 0.999
28.18 1.30 1.32 330 0.999
29.60 0.669 0.661 550 0.999
a v ) a p exptp + bexp; aexp, slope; bexp, intercept; ath, theoretical acceleration; ttop, time of flight of primary insulin ions.
part of the plume (t). First, the values of vp corresponding to the maxima of the MS peaks were plotted versus tp to confirm constancy of the acceleration (Figure 5). The slope of each line, aexp, represents acceleration and can be compared with the theoretical value of acceleration, ath ) ze(Va - Vb)/(ms) in Table 2. The linearity of the graphs and agreement between the measured and calculated accelerations are satisfactory. Electric field strengths in the extraction region are not significantly distorted 350 ns after the pulse of the desorption laser. The value of the intercept does not necessarily indicate the most probable initial velocity of the insulin ions because desorption and ionization processes are much more complex at the beginning. This is most striking in the case of negative values of the intercept because the initial velocity cannot be negative. The energy difference, ∆E, provides additional information about the plume (Figure 6). It was evaluated according to eq 5 from linearized data for all experimental conditions in the region t, where the ion intensity was above 50% of the intensity at the peak maximum. Each of the six groups of plots reveals the energy spread within the center of a plume generated at a specific voltage, Vb. The three lines within each group correspond to three different values of xp, and they should be identical under ideal conditions. There is no apparent order among the lines within a group, which would point to energy loss between the three photodissociation locations due to collisions. The discrepancies stem, most likely, from our inability to determine the geometrical parameters with sufficient accuracy and precision. For example, an inaccuracy of 0.1 mm in the determination of xp can lead to an
Figure 6. Calculated energy difference ∆E within the mid-range of insulin peak at three different positions of the photodissociation spot, xp ) 1.0 (solid); 2.6 (dotted), and 3.8 mm (dashed). The six groups correspond to ions accelerated under the same conditions as in Figure 2.
error of 120 eV at the highest extraction field strength. Despite the large differences between the lines within each group, the main trend is apparent. The plumes generated at high electric field strengths show energy deficit and large energy distributions. On the other hand, the plumes generated at low electric field strengths show less dramatic energy distributions and even energy excess. The energy offset and distribution can stem from the interaction of insulin ions with matrix neutrals. This will be explained in more detail in the Conclusion section. The last step in the data analysis includes evaluation of the initial distributions of ion velocity, location, and time of creation within the particular plumes. The parameters v0, x0, and t0 were calculated from eqs 2, 6, and 7, or eqs 8 and 9 from linearized tp and t within the peak region with intensity higher than 50% of the peak maximum for each xp and Vb. The first equation set was used for the evaluation of the movement of energy-deficit ions. One of the equation sets was then used to solve for the movement of the ions with an excess of energy. The spread of v0, x0, and t0 within the evaluated region for Vb ) 0.0 kV is shown in Figure 7. One can immediately notice the differences among the solutions of data taken at different values of xp and the improbable shape of the initial velocity curve. The discontinuities on the curves separate the regions where the first (longer t, x0 > 0) or the second (shorter t, x0 ) 0) equation set was used. The values of x0 are below 0.2 mm, which is comparable to the accuracy in determination of TOFMS dimensions. However, Figure 7 shows reasonable values for the parameters. The difference between the solutions obtained for the data taken at the three positions of the photodissociation locations is even larger for Vb ) 14.0 kV (data not shown), and these cannot be explained as a result of geometric inaccuracies. The results suggest that MALDI is more complex a process than can be adequately described by either of the two simple equations sets. Still, a comparison of the results obtained for Vb ) 0.0 and 14.0 kV provides a rough picture about the differences between the temporal and spatial scales of the two events. At large field strength, the insulin ions are formed within 0.1 mm from the surface shortly after the desorption pulse. Because both x0 and t0 are small, velocity calculation is unreliable. The unreliability is also indicated by a sharp minimum on the velocity curves (due to different sets of equations used for the
Figure 7. Initial velocity, spatial, and temporal distributions calculated for different parts (time of flight) of the plume at high extraction field strength, Vb ) 0.0 kV, from data obtained at three xp values (same designations as in Figure 6).
Figure 8. Temporal evolution of the plume of matrix neutrals obtained at xp ) 5.0 mm by LD-LPI from a sample film of ferulic acid and insulin. MS signals are plotted as the peak heights of the matrix ion [M - 17]+.
calculation of the front edge and for the tail of the insulin peak). At low field strength, the distributions of x0 and t0 obtained for the three values of xp differ significantly and have larger values. This points to additional phenomena, such as collisions of the insulin ions. The same experimental arrangement was also used to evaluate the flux of matrix molecules. Here, the second laser photoionizes the matrix neutrals or fragments. The method is commonly known as laser desorption-laser photoionization (LD-LPI). The ions produced by 193-nm photoionization of matrix are the same as those from 337-nm laser desorption, [M + H]+ and [M - 17]+. The plot of the peak height of the lighter ion vs tp at constant xp ) 5.0 mm shows long tailing of the plume (Figure 8). The velocity at the front edge of the matrix plume is as high as 1500 m/s. Most ions, however, move at velocities on the order of 100 m/s. The photoionization laser ionized a ∼100-µm slice of the matrix Analytical Chemistry, Vol. 69, No. 21, November 1, 1997
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plume. So, the velocity range of irradiated matrix molecules increases with longer tp. The tailing may also be caused by the temporal spread in the desorption of matrix neutrals. Regardless of which is the dominant reason, there is always a large overlap of ejected matrix molecules with the plume of insulin ions. Insulin ions generated at high electric field strengths will be quickly accelerated above 1500 m/s and will escape the plume of the matrix. The lower the electric field strength, the more collisions between an insulin ion and matrix neutrals will occur to slow down the ions. CONCLUSION The MALDI-LPD method enabled us to create a hole in the plume of insulin ions, which was detected as a negative spike in the mass spectrum of insulin. Because the hole could be created in the plume soon after the desorption pulse, interesting information about the early stages of the MALDI process were obtained. Comparison of the ion kinetic energy with decrease of its potential energy in the electric field revealed an energy difference. The energy difference can be negative (energy deficit) or positive (energy excess), and it can change within a single plume. While energy excess can be explained only by a positive initial velocity, energy deficit can stem from several reasons. We excluded effects of electrical shielding in the extraction region. Space-charge effect was not a probable cause either. Two explanations for the energy spread remain: initial velocity or spatial variation of the insulin ions, or both. Our efforts to solve a set of equations to obtain complete solutions for the velocity, spatial, and temporal distributions were partially successful. The discrepancies between theory and experiment are due to either insufficient accuracy in determining the input variables (especially geometrical parameters of the TOFMS and various voltages) or complex movement of the desorbed ions and molecules. Both molecules and ions undergo many collisions during the initial period. This is consistent with the differences found between the energy profiles of the plumes of insulin ions desorbed at different extraction voltages. The desorption laser ejects a plume of matrix and analyte species. Most of the matrix is desorbed in the neutral form, and its production probably continues for a period longer than the duration of the desorption pulse. Most of the matrix neutrals move faster than analyte neutrals, but tailing of the velocity distribution of matrix molecules ensures an overlap with the plume of insulin. Most insulin ions are formed shortly after the desorption pulse, directly in the film or within a distance of a few hundred micrometers while the desorbed plume is dense. The insulin ions start to move through the plume of the matrix neutrals being accelerated by the electric field. At high electric field strengths, the ions that are created first escape the plume earlier and do not lose much of their initial kinetic energy. The ions created later have to travel farther in the plume of unaccelerated matrix neutrals that passed them in the meantime. These undergo more collisions and lose more
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kinetic energy than the insulin ions created earlier. The overall energy deficit of the later-created ions may be further promoted by a lower initial velocity and by ion formation farther away from the repeller plate. The ions within a plume generated at low electric field strength show less dramatic differences in kinetic energy. The accelerating force of the electric field is weaker, and ions spend a longer time in the matrix plume. All insulin ions undergo many collisions, and their velocities will match more closely the velocity of the matrix molecules. Matrix molecules are also moving, so they slow down only the fastest analyte ions but help to accelerate the slower insulin ions. Therefore, the entire plume may exhibit energy excess at low electric field strengths. Plumes desorbed at high extraction field strengths are highly organized, i.e., a specific part of the plume was created in a specific position at a specific time with a specific velocity, and the distributions were not randomly altered by collisions. The existence of such a correlation between initial ion distributions of different types is advantageous if one wants to introduce additional operations to cancel the effects of broadening. This is why a high electric field is beneficial whether employing timedelay extraction or an ion mirror to focus the ions. Our results are especially valuable for the design of time-delay extraction, because it enables characterization of ion motion in the extraction region on a submicrosecond time scale. Knowledge of vp, xp, and tp will be useful for the proper design of extraction pulse shape. In cases of well-organized plumes desorbed at high electric field strengths, the application of a precisely tailored pulse shape rather than a rectangular pulse should result in further increases in resolution. In fact, the employment of LPD itself may be useful for improving the resolution within a limited mass region. The resolution of the spike is about 5 times higher than that of the original insulin peak in our case. The photodissociation laser irradiates only a thin “slice” of the plume. Therefore, ions with narrower velocity, spatial, and temporal spreads are selected. Much larger improvement of the resolution should be achievable with the use of a well-focused picosecond photodissociation laser. ACKNOWLEDGMENT We thank Dr. Ho-ming Pang for lending us the digital-delay generator. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. This work was supported by the Director of Energy Research, Office of Basic Energy Sciences, Division of Chemical Sciences. Received for review March 5, 1997. Accepted August 21, 1997. AC970248F X
Abstract published in Advance ACS Abstracts, October 1, 1997.
