Properties of Matrix-assisted Laser Desorption. Measurements with a Time-to-Digital Converter W. Ens,* Y. Mao, F. Mayer and K. G. Standing Department of Physics, University of Manitoba, Winnipeg, Canada R3T 2N2
SPONSOR REFEREE: Dr Brian Chait, The Rockefeller University, New York, USA
Some properties of matrix-assisted laser desorption have been studied using single-ion-counting methods and a time-to-digital converter. The methods allow examination of the process for irradiances near the reported threshold for observation with a transient recorder. All measurements were made using bovine insulin as a test compound. We present direct evidence that an irradiance threshold near lo6W cm-* exists for ion production, and that the process is a collective effect, either involving a large number of molecular ions ( - 10‘) in a successful event or none at all. Above the threshold, the yield is found to scale with a high power (4th to 6th) of the irradiance. Measurements of initial velocity distributions indicate an axial velocity spread corresponding to 50 eV and a radial velocity spread corresponding to 2.4 eV. Thus the ejection or extraction mechanism appears to be strongly asymmetric.
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The introduction of matrix-assisted laser desorption (LD) by Tanaka et al.’ and Karas and Hillenkamp,’ as a method of producing intact molecular ions from proteins and peptides, has significantly extended the mass range accessible to mass spectrometry. In the technique of Karas and Hillenkamp, the sample is mixed in solution with a suitable matrix (originaly nicotinic acid) in molar proportions of about 1:1000 and dried onto a sample foil. UV laser pulses (originally 266nm) with irradiance of about lo6 to lo7W cm-’ strike the target releasing large numbers of intact molecular ions of the sample. The ions are analysed in a time-of-flight spectrometer with an electron multiplier for detection. To date all the experiments have used a transient recorder to record the data, i.e., they use a current measurement. Spectra from single shots are normally summed together at a repetition rate of about 1Hz. In the last 2 years, considerable progress has been made in the field. A number of new matrices have been discovered3 and the range of suitable wavelengths has been greatly increased.”6 In addition the quality of spectra has improved steadily. In particular, it has been shown that the signal-to-background ratio and the resolution improve as the power density is reduced to a ‘threshold’ i r r a d i a n ~ e . ~ The threshold level depends on the apparatus used for the measurement. Because a transient recorder requires a large number of ions per pulse to obtain useful data in a reasonable period of time, it is difficult to deduce what happens in the neighbourhood of the threshold; especially because the shot-to-shot reproducibility is rather poor. Two descriptions of the desorption process are consistent with the observed ‘threshold’: (i) the desorption could be a collective effect in which any successful desorption event produces a large number of molecular ions. Near the threshold irradiance, there is a precipitous decrease in the probability for producing a desorption event from a given laser pulse, but the number of molecular ions in a given successful event is still large. (ii) On the other hand, the threshold could also be explained by a drastic decline in Author to whom correspondence should be addressed.
the number of ions produced per desorption event, with a consequent decrease in measurement efficiency. In this description, most of the laser pulses continue to produce molecular ions near the irradiance threshold , but the average number of ions produced becomes smaller. If the latter explanation is correct, it seems likely that greater sensitivity could be obtained in the neighborhood of the threshold by using a time-todigital converter (TDC) instead of a transient recorder. The TDC gives a single output pulse for each event detected whose amplitude is larger than a specified value, so it may be optimized for observation of a small number of ions per laser event. It may also be run at a high repetition rate, yielding useful statistics even if the number of events per pulse is low. Here we report the first matrix-assisted LD measurements using single-ion counting. The objective of these experiments was to resolve the question of whether the desorption process is a collective effect. Is it possible to initiate desorption events in which single or very few molecular ions are produced, or is the minimum number of molecular ions per event very large? In addition to improving our understanding of the mechanism, the experiments were motivated by the possibility of improving the spectral quality and increasing the sensitivity if the observation threshold could be lowered. EXPERIMENTAL The schematic arrangement of the experiment is shown in Fig. 1. Here, laser pulses of about 10-2011s in duration from a high-repetition-rate excimer laser (HE460-HR-B) (Lumonics Inc. , Kanata, Ontario, Canada) at 308 nm are incident on the target surface at 45” to the normal. The laser spot size is focused onto an area approximately 50 pm by 80 pm as measured by removal of nitrocellulose from an aluminum target. The power density is controlled in reproducible steps between lo6 and lo7W cm-*, by a series of UV fused silica plates with 8% attenuation per plate. The energy per pulse was measured with a Model 365 power and energy meter (Scientech Inc., Boulder, CO, USA).
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57 Computer
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Deflection Plates Figure 1. Schematic diagram of the experimental arrangement. Focusing optics are omitted for clarity. The deflection plates are about 30 cm from the target, and the detector is movable between 40 and 140cm from the target.
Just before entry to the mass spectrometer, the beam passes through a displacement plate mounted on a stepping motor to allow the beam to be rastered across the target. The time-of-flight spectrometer (TOF I, Manit~ba',~), constructed in-house is a simple linear instrument with a single acceleration region (5 cm in this case), a field-free flight path (variable from 40 to 140 cm") and a chevron microchannel plate detector. A variable diameter iris diaphragm is mounted in front of the detector to control the acceptance. Ions are accelerated by applying a potential (typically 14 kV) to the target. The front plate of the detector is set to approximately - 2 kV. The beam can be deflected in 2 perpendicular directions by deflection plates situated 40 cm from the target. The plates are 5 cm square and separated by 3cm. Low-mass ions may be suppressed by pulsing the deflection plates in the field-free region. Amplified signals from the microchannel plates can be observed directly on an analogue oscilloscope, or fed into a 255-stop TDC (model CTN-M2 from Institut de Physique NuclCaire, Orsay, France) connected by a custom interface to an Atari Mega ST computer for data storage and analysis. Bovine insulin was selected as a test compound. Approximately 0.1 g/L (or less) insulin and 10 g/L sinapinic acid were dissolved in 0.1% TFA in water and acetonitrile (1: 4 by volume). Approximately 20 pL of this solution was dropped onto about 3cm2 area of etched silver foil and allowed to dry. The resulting sample thickness was found to give optimum reproducibility for our purposes; it is a few times thinner than that typically used by other Because of the steep dependence of the yield on irradiance, we anticipated that conditions under which single (or a few) ions are produced from a single laser
pulse would be extremely critical (if they existed), and that finding such conditions would be easier starting from high intensity where the experiment was known to work. A TDC (even a multi-stop device) can only record one count in a given time period determined by the dead-time (here 200 ns). In practice this means that no more than one ion of a given mass can .be detected for each laser pulse. If a large number of ions of a given mass strike the detector at the same time, only one pulse is recorded, so no information about the intensity of the signal is obtained. Therefore the procedure outline below was followed in order to get meaningful data with the TDC for irradiances where many ions are produced from a single pulse, as well as for lower irradiance. Starting with an irradiance where many ions of the same mass are produced in a single event (i.e., an irradiance where the detector pulses could be observed on an oscilloscope with the iris fully open), the iris was closed down so that on the average less than one ion per laser pulse passed through the aperture. In that situation, the probability for detection is related to the total ion yield, i.e., it is independent of whether the ions appear in many pulses with only a few ions per pulse, or whether they appear in a smaller number of pulses with more ions per pulse. The average intensity of the signal from a single laser pulse is then represented by the total number of detected ions after many laser shots. With at most one count registered per event for a given mass, a high repetition rate is required to get useful statistics. The excimer laser is capable of running up to 500 Hz but for the present experiment it was not necessary to go beyond 40Hz. A spot on the sample becomes exhausted after about 100 laser shots so it was necessary to move the beam across the target with the displacement plate mentioned above to expose fresh sample to the laser. The beam was rastered over a 5mm square at the centre of the target allowing an array of 40 by 40 spots to be irradiated without overlap. Typically a spectrum could be acquired by irradiating one row of 40 spots. The etched silver targets were selected because the solution spreads evenly over the surface giving reproducibility within about 20% for adjacent rows and within about 50% over the 5mm square examined. When comparisons between many spectra were made, control spectra were run on adjacent rows for normalization. Blocking part of the secondary beam, and rastering the laser is necessary to obtain data in the high irradiance region. In addition, it allows averaging over a large number of laser shots and over a relatively large part of the target, giving considerably better reproducibility than is normally available. Thus the method is useful for the fundamental studies reported here although there may not be any immediate analytical advantages over the standard matrix-assisted LD experiment.
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RESULTS AND DISCUSSION 1. Mass spectra by single-ion counting As a first step toward using a TDC to measure LD spectra, the power density was set just above the threshold for observation of the molecular ion signal on an oscilloscope with the iris open. The iris was then
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closed down to a point where the average number of counts for the insulin molecular ion was less than one per laser pulse. With this irradiance, reasonable statistics were obtained after about 1600 shots on 40 different spots; i.e., 40 shots per spot. The spectrum of bovine insulin obtained in this manner is shown in Fig. 2(a). The deflection plates shown in Fig. 1 were pulsed to suppress low-mass background. As the irradiance is decreased from this level, more spots are required to get useful statistics, or alternatively, the iris diameter can be increased (see below). To illustrate the effect of reducing irradiance, the spectrum in Fig. 2(b) was taken with the same iris diameter but about 1/3 the irradiance used in Fig. 1;the spectrum was accumulated for 8000 laser shots. As reported earlier,7 there is a clear improvement in resolution for the molecular ion, and the Iow-mass background is decreased as the irradiance is decreased. The resolution (mlhm, full-width at half-maximum) obtained at this power density is about 500. This is similar to the resolution reported by Beavis and Chait7 but not significantly better, as might be expected if our irradiance was lower. However, a direct comparison of resolutions is difficult because the conditions are not the same; in particular, the large acceleration region (5 cm) in our spectrometer will reduce resolution significantly if the initial axial velocity spread gives a significant contribution; see below. 2. Irradiance threshold
The results in Fig. 2 indicate an improvement in the spectrum as the irradiance is reduced. However at the lower irradiance the count rate is very low; this may be increased by opening the iris. To determine how far the irradiance could be reduced, the insulin molecular ion yield was measured as a function of irradiance for several different iris diameters. If the threshold for observing molecular ions by laser desorption was instrumental (i.e., related to detection) then it would be expected to decrease for larger iris diameter, since more ions would strike the detector, giving higher probability for detection. The yield increases with the iris diameter as expected (see later discussion and Fig. 6), but to illustrate the variation of yield with irradiance in Fig. 3, we have
"? I 40
[M+H]+ I
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Flight Time (ns) Figure 2. Positive-ion mass spectra of bovine insulin with power densities (a) 6 x lobW cm-', and (b) 2 X lo6W cm-'. For both spec-
tra, light ions were suppressed by pulsing the deflection plates shown in Fig. 1.
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Power Density (W/cm2) Figure 3. Dependence of the insulin molecular-ion yield on irradiance for several different iris diameters: 0 1.5 mm, 3.0 mm,
05.0 mm, A 7.0 mm. The 3 lines indicate scaling with the 4th, 5th and 6th power of the irradiance.
normalized the data for different iris diameters at 3.1 X lo6W cm-2. Different symbols represent different iris diameters. The point on the horizontal axis is an upper limit since no peak was observed there but all the other points represent a detected insulin yield. In each case no insulin signal was observed after the addition of one more attenuator. It is clear from these data that regardless of the iris diameter, the signal disappears at about the same irradiance within about 8% (the attenuation of one plate). Thus the data indicate a true irradiance threshold near 2 X lo6W cm-' for the production of molecular ions. This value of the threshold is only approximate since there is both a temporal and a spatial distribution of deposited energy, neither of which is accurately known; the estimate of 2 X lo6W cm-' was made assuming uniform distributions. It should be noted that our measurements refer only to production of ions, not neutrals. However, the existence of a threshold is predicted by calculations of the total ejecta (ions and neutrals) based on Beer's Law both for thermal ablation" and for a pressure-pulse model. l2 The adiabatic absorption model introduced by Vertes et al. ,I3 also predicts a threshold irradiance for the onset of plasma ionization. Above the threshold irradiance, for at least a small region, the yield increases rapidly, scaling approximately with the 4th to the 6th power of the irradiance. The 3 lines drawn on Fig. 3 indicate the 4th, 5th and 6th power of the irradiance. A 5th power dependence on irradiance has been reported previously for the desorption yield of neutral molecules of tryptophan. l4 3. Collective effects The measurements described above indicate that the total number of ions produced decreases sharply with decreasing irradiance, but do not show how these ions
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are distributed from pulse to pulse. At one extreme, the number of ions desorbed per event might decrease uniformly. At the other extreme, the number of ions per successful desorption event might be constant, with the probability for such an event decreasing with decreasing irradiance. To resolve this question pulseheight distributions were measured at the threshold irradiance for different iris diameters. The pulse-height is a measure of the number of ions that strike the detector. If the number of ions per successful event is large (but the number of successful events is small), then the pulse-height will increase as the iris is opened, since more ions will strike the detector. If the number of ions per event is small but constant, then the pulseheight will remain the same as the iris is opened because, even for an open iris, the average number of detected ions per incident pulse is less than one. The simplest method of determining the pulse-height distributions is to increase the discriminator level. Only pulses higher than the discriminator level are registered in the TDC; for an increment in the level from V to (V-tdV), the decrease in counts corresponds to the number of pulse-heights in that interval. The results are shown in Fig. 4: as the iris diameter is increased, the discriminator level required to reduce the signal becomes larger indicating that the pulse-height from the detector is increasing. For the 25 mm iris, there is no loss of signal until the discriminator level is >200mV. Thus it appears that there is a minimum number of molecular ions in a successful event; i.e., it is a collective effect. Because there is a high ratio of matrix to sample in the substrate, and because the technique is largely insensitive to the protein species15,the collective effect almost certainly depends only on the matrix, i.e., a minimum number of matrix ions must be desorbed in a successful event. Therefore it should be possible to reduce the number of sample ions desorbed per event by reducing the concentration of the sample molecules in the solution. The result of reducing sample concentration by factors of 10 and 100 are shown in Fig. 5. The molecular ion signal drops as expected, as does some of the background, but the signal-to-background ratio becomes progressively worse, suggesting that a significant part of the background has its origin in the matrix. Reduction of the sample concentration by another factor of ten gave no detectable molecular-ion signal
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Figure 5. Mass spectra of insulin for 3 different sample concentrations: top, 0.1 g/L; middle 0.01 g/L; bottom, 0.001 g/L. The matrix concentration is 10 g/L in each case and 20 pL of the solution is spread over an area of 1 cm'.
above background. Still, it appears to be possible to acquire useful spectra with significantly reduced sample concentration. In order to estimate the minimum number of molecular ions associated with a successful event, for a given sample concentration, the yield was measured as a function of iris diameter. As long as the average number of ions passing through the aperture for a successful event is much less than one, the measured yield should increase linearly with the area. Once the average number of ions passing through the aperture exceeds the number required for near 100% detection efficiency, every successful event will be detected and the measured yield will saturate. For the lowest sample concentration (0.001 g/L), such a saturation is observed for an iris diameter of about 7mm (Fig. 6(a)). The number of counts measured in a region of low background continues to increase linearly beyond this aperture. Thus there is at least one insulin ion in an area of diameter 7 mm. From measurements of the radial velocity distribution (see below) the ions are distributed over an area of diameter 36 mm for the present field-free path length of 140cm. A rough estimate of the total number of molecular ions that could be detected is then (36/7)' or a few tens of ions. The actual ion yield may be greater since the detection efficiency for a single insulin ion with energy of 16 keV is likely to be considerably less than 100%. The saturation occurs at about 3 mm diameter for the 0.01 g/L sample concentration as shown in Fig. 6(b). This corresponds to several hundred ions per event. For a concentration of 0.1 g/L the yield saturates very close to the minimum iris opening suggesting that 103-104molecular ions are ejected per pulse. It is possible to estimate a charge-to-neutral ratio for the ejected species based on the number of ions ejected from the target, and on the amount of sample removed from the surface. Two assumptions are needed for the estimate to be meaningful: (i) the loss of the molecularion signal due to repeated laser pulses corresponds to the removal of the sample from the irradiated area. Examination of the targets under a microscope after irradiation is consistent with this assumption. (ii) Any
120 RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL 5. NO 3, 1991
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removed sample that does not appear as molecular ions, is ejected as intact neutral molecules; i.e., fragments and clusters (including macroscopic chunks) do not account for a significant amount of the removed material. Identifiable charged fragments have not been observed so this assumption is plausible. There is a signal from negative molecular ions about the same intensity as the positive-ion signal, and a much smaller signal from multiply charged species and from cluster ions. The charge-to-neutral ratio is calculated for 20 pL of 0.1 g/L insulin solution deposited onto 3 cm2 area, or about 1 pmol/mm2, and a laser irradiance near threshold. Under these conditions, the molecular-ion signal disappears after about 100 laser shots as indicated in Fig. 7. Since the beam spot is -0.005 mm2, it appears that 4 x lo7 molecules are removed per laser shot. If the number of molecular ions ejected is -lo4, then there is an excess of neutrals by a factor greater than lo3. A similar charge-to-neutral ratio is observed for particle-induced desorption. l6
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Detector Iris (cm2) Figure6. Insulin yield (0)measured as a function of detector-ins diameter. The squares represent background counts measured in a low-background region. (a) Sample concentration 0.001 g/L; (b) sample concentration 0.01 g/L. The matrix concentration was 10 g/L and 2 pL of the solution was deposited on an area of 1cm2.
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Number of shots per spot Figure7. Measured yield for insulin as a function of the number of laser shots per spot. The yield saturates indicating that the signal from a given spot disappears after about 100 shots.
4. Initial velocity distributions Both the radial and axial velocity distributions have an important influence on instrument design and performance. The radial velocity distribution (along with the acceleration voltage), determines the solid angle of ejected ions, and therefore the transmission of the instrument for a given geometry. In the present experiment, the acceptance is particularly important in estimating the total ion yield. The axial velocity distribution and delays in the desorption process or in the extraction of ions affect the resolution and calibration of the spectra. Measurements of the dynamics of the ejection process also contribute to the understanding of the desorption mechanism. Radial uelocity. Radial velocity distributions were measured in 2 perpendicular directions using deflection plates in the field-free region of the spectrometer. Yields of insulin were measured as a function of deflection voltage with an iris diameter of about 1mm for the maximum field-free drift length of 140 cm. The distributions are shown in Fig. 8. From the geometry and the acceleration voltage, the radial velocity may be calculated for ions detected at a given deflection voltage. For the distributions in Fig. 8, the points at half intensity represent ions with a radial velocity u,, corresponding 2.4 eV; the fringing fields of the deflection to mu:/2 plates were taken into account for these calculations. Ions with this radial velocity would strike about 18mm from the centre of the detector if no deflection voltage were present. Thus most of the ions appear within a circle of diameter 36 mm. For a uniform distribution of ions spread over a 36mm diameter circle, a 1mm diameter aperture samples approximately 0.1% of the ions. The width of the radial velocity distribution is comparable to that of insulin ions ejected by keV or MeV particle b~mbardrnent.’~ The radial-velocity distributions for ions from laser desorption appear to be the same for molecular ions and low-mass ions and presumably correspond to emission centred about the target normal. This is also the situation for low-energy bombardment, but is in contrast to MeV bombardment where large molecular ions have radial velocity distributions correlated to the incoming ion direction (although light ions show no such correlation). Axial uelocity. In order to obtain information on the initial axial velocity ( u , ~ )distributions, we measured
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the peak width as a function of path length. This measurement is considerably less direct than the measurement of the radial velocity since there are several possible contributions to the width of the peak; the time-width of the laser pulse, the axial velocity distribution and possibly a spread in the time of emission or extraction. The results for an irradiance of about twice the threshold value are plotted in Fig. 9. The fact that the width increases with flight-path (plotted as flighttime) indicates that an axial velocity spread accounts for at least part of the broadening. The axial velocity spread may be extracted directly from the slope of a line through the data. The 3 lines shown in Fig. 9 are calculated peak widths for 3 axial velocity spreads, indicating that the spread in mu:,/2 is 50 eV for the insulin molecular ion. This result is consistent with that of Beavis and Chait.ls The intercepts of the calculated lines in Fig. 9 correspond to fixed time-spreads which include at least the time-spread during acceleration and the laser-pulse duration. The time-spread during acceleration caused by the axial velocity spread Au,, depends also on the average initial velocity u,,. Because of the large acceleration region (5 cm) in our spectrometer, this spread makes an appreciable contribution to the final peak width. If a very simple picture of ion ejection and extraction is adopted, i.e., if the ions are extracted at the same time from an equipotential plane, then an average initial axial velocity can be extracted from the data. The interce ts of the 3 lines shown in Fig. 9 P 40, 70 and 120 eV, suggesting correspond to muZo/2 that in this description the average initial axial velocity is comparable to the spread in axial velocity.
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120- Y-deflection 100-80-60-40-200--~~1.~~1~ -600-400-200 0 200 400 600
-600-400-200 0 200 400 600
Deflection Voltage (volts) Figure 8. Yield of the insulin molecular ions measured as a function of deflection voltage. The number of ions detected drops to zero rapidly for a deflection voltage greater than 150 V. Ions detected with this deflection voltage have a radial velocity u, where mu:/2 2.4 eV.
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2401
I100 Figure 9. Insulin peak width measured for different field-free path lengths at constant accelerating voltage. The 3 lines represent the calculated peak width considering only the laser-pulse duration and the initial axial velocity: (a) Uo= 40 eV, AUo= 40 eV; (b) Uo= 70 eV, AUo=50eV, (c) Uo=120eV, AUo=60eV; where Uo=muL/2.
Both the Rockefeller group18 and the Uppsala group" have shown evidence for a spread in emission time as well as in the initial velocity. The data of Fig. 9 can be accounted for without considering such a spread. On the other hand, a more complex process involving time- or spatial-spreads in emission or extraction of the ions cannot be excluded by the data. Such contributions would not affect the slope of the calculated line, so the estimate of the axial velocity spread would be unchanged. However, a different mean initial axial velocity would have to be assumed to fit the data. An axial velocity spread corresponding to 50 eV and a radial velocity spread corresponding to about 3 eV indicates a strongly asymmetric ejection process. The targets used in this experiment were deliberately rendered coarse from acid etching so, on a microscopic scale, there is no well-defined normal direction. The difference in the radial and axial velocity spreads therefore suggests that the process averages over a macroscopic region obscuring the detailed structure. This is consistent with the suggestion that a rapidly expanding macroscopic gas jet is released from the target.6."* 2o An asymmetry between the radial and axial velocity spreads can also be explained by considering the orientation of the acceleration field. If ions are not accelerated by the field until the density of the gas plume is sufficiently low, there will be a spread in the final energy of the ions corresponding to a spread in the distance (2) from the target where unimpeded extraction begins." Such a spread would not affect the radial velocity, but would cause a defect in the axial velocity corresponding to longer flight-times. This explanation is also consistent with observations of the peak shape for increasing irradiance. As shown in Fig. 10, the broadening of the molecular ion peak for insulin with increasing irradiance occurs entirely on the high-time side. The same effect has also been reported by the Uppsala group," but in that case the broadening was much more pronounced for the same increase in irradiance. The difference in the amount of broadening may be understood qualitatively by the fact that the acceleration distance in the present experiment is about 5 times larger than in the Uppsala experiment, reducing the significance of a spatial spread.
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Acknowledgements This work was supported by grants from the US National Institutes of Health (Institute of General Medical Sciences), and from the Natural Sciences and Engineering Research Council of Canada. One of us (F.M.) gratefully ackowledges partial support from the Deutsche Forschungsgemeinschaft. ) I
REFERENCES
v)
1. K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida, Rapid Commun. Mass Spectrom. 2 , 151 (1988). 2. M. Karas, D. Bachmann, U. Bahr and F. Hillenkamp, Int. i. Mass Spectrom. Ion Processes 78, 53 (1987); Anal. Chem. 60, 2299 (1988). 3. R. C. Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3,432 (1989). 4. R. C. Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3, 436 (1989). 5. A. Overberg, M. Karas, U. Bahr, R. Kaufmann and F. Hillenkamp, Rapid Commun. Mass Spectrom. 4, 293 (1990). 6. R. W. Nelson, R. M. Thomas and P. Williams, Rapid Commun. Mass Spectrom. 4, 348 (1990). I . R. C. Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3, 233 (1989). 8. B. T. Chait and K. G. Standing, Int. 1.Mass Spectrom. Ion Phys. 40,185 (1981). 9. K. G. Standing, R. Beavis, G. Bolbach, W. Ens, F. Lafortune, D. Main, B Schueler, X. Tang and J. B. Westmore, Anal. Instrum. 16, 173 (1987). 10. B. Schueler, R. Beavis, W. Ens, D. E. Main, X. Tang and K. G. Standing, Int. .I M . ass Spectrom. Ion Processes 92, 185 (1989). 11. R Srinivasan and B. Braren, Chem. Reu. 89,1303 (1989). 12. R. E. Johnson, in Ion Formation From Organic Solids (IFOS V ) , ed. by A. Hedin et al., John Wiley and Sons, Chichester, p. 189 (1990); R. E. Johnson, S. Banerjee, A. Hedin, D. Fenyo and B. U. R. Sundqvist, in Methods and Mechanisms for Producing Ions From Large Molecules, in the NATO AS1 Science Series, ed. by W. Ens and K. G. Standing, Plenum Press (in press). 13. A. Vertes, M. De Wolf, P. Juhaszand R. Gijbels, Anal. Chem. 61, 1029 (1989); A. Vertes, L. Balazs and R. Gijbels, Rapid Commun. Mass Spectrom. 4, 263 (1990). 14. B. Spengler, U. Bahr, M. Karas and F. Hillenkamp, Anal. Instrumen. 17, 173 (1988). 15. R. C. Beavis and B. T. Chait, Proc. Natl Acad. Sci. USA 87, 6873 (1990). 16. M. Salehpour, P. Hiikansson, B. Sundqvist and S. Widdiyasekera, Nucl. Instr. Meth. B13,278 (1986). 17. W. Ens, B. U. R. Sundqvist, Per Hlkansson, A. Hedin and G. Jonsson, Phys. Reu. B39, 763 (1989); W. Ens, B. U. R. Sundqvist, P. Hlkansson, D. Fenyo, A. Hedin and G. Jonsson, 1.Physique. C2, 9 (1989). 18. R. Beavis and B. T. Chait, in Methods and Mechanisms f o r Producing Ions From Large Molecules, in the NATO AS1 Science Series, ed. by W. Ens and K. G. Standing, Plenum Press (in press). 19. A. Hedin, A. Westman, P. Hiikansson and B. U. R. Sundqvist, ibid. 20. P. Williams and R. W. Nelson, ibid. 21. M. Karas and F. Hillenkamp, in Advances in Mass Spectrometry, Vol. II, ed. by P. Longevialle, Heyden and Son, London p. 354 and 416 (1989).
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6XIOO
Figure 10. Insulin molecular-ion peak for four different irradiances ranging from threshold to about 3 times threshold irradiance. The data are displayed in 32 ns bins smoothed over 7 bins; peak heights are normalized. The peak width increases monotonically with irradiance, all of the broadening taking place on the high-time side.
CONCLUSION We have demonstrated that there is a true irradiance in matrix-assisted laser threshold near lo6W desorption and that the effect is a collective one involving a minimum number of sample ions. This was suggested after the initial discovery of the phenomenon:” but until now the evidence has been indirect. In a typical matrix-assisted laser desorption measurement of bovine insulin, the number of positive molecular ions ejected is lo4. Assuming that the sample is removed when the signal disappears, and that it is ejected as intact molecules, the number of neutrals exceeds the number of ions by a factor of lo3to lo4.The number of molecular ions ejected in one pulse can be reduced by reducing the sample concentration; useful spectra can be obtained with ejection of a few tens of ions. Measurements of initial velocity distributions for bovine insulin indicate a spread in the axial velocity corrsponding to 50 eV. The spread in radial velocity is much smaller; about 2.4eV, thus indicating a strongly asymmetric ejection or extraction mechanism.
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Received 12 January 1991; accepted 13 January 1991.
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SPONSOR REFEREE: Dr Brian Chait, The Rockefeller University, New York, USA
Some properties of matrix-assisted laser desorption have been studied using single-ion-counting methods and a time-to-digital converter. The methods allow examination of the process for irradiances near the reported threshold for observation with a transient recorder. All measurements were made using bovine insulin as a test compound. We present direct evidence that an irradiance threshold near lo6W cm-* exists for ion production, and that the process is a collective effect, either involving a large number of molecular ions ( - 10‘) in a successful event or none at all. Above the threshold, the yield is found to scale with a high power (4th to 6th) of the irradiance. Measurements of initial velocity distributions indicate an axial velocity spread corresponding to 50 eV and a radial velocity spread corresponding to 2.4 eV. Thus the ejection or extraction mechanism appears to be strongly asymmetric.
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The introduction of matrix-assisted laser desorption (LD) by Tanaka et al.’ and Karas and Hillenkamp,’ as a method of producing intact molecular ions from proteins and peptides, has significantly extended the mass range accessible to mass spectrometry. In the technique of Karas and Hillenkamp, the sample is mixed in solution with a suitable matrix (originaly nicotinic acid) in molar proportions of about 1:1000 and dried onto a sample foil. UV laser pulses (originally 266nm) with irradiance of about lo6 to lo7W cm-’ strike the target releasing large numbers of intact molecular ions of the sample. The ions are analysed in a time-of-flight spectrometer with an electron multiplier for detection. To date all the experiments have used a transient recorder to record the data, i.e., they use a current measurement. Spectra from single shots are normally summed together at a repetition rate of about 1Hz. In the last 2 years, considerable progress has been made in the field. A number of new matrices have been discovered3 and the range of suitable wavelengths has been greatly increased.”6 In addition the quality of spectra has improved steadily. In particular, it has been shown that the signal-to-background ratio and the resolution improve as the power density is reduced to a ‘threshold’ i r r a d i a n ~ e . ~ The threshold level depends on the apparatus used for the measurement. Because a transient recorder requires a large number of ions per pulse to obtain useful data in a reasonable period of time, it is difficult to deduce what happens in the neighbourhood of the threshold; especially because the shot-to-shot reproducibility is rather poor. Two descriptions of the desorption process are consistent with the observed ‘threshold’: (i) the desorption could be a collective effect in which any successful desorption event produces a large number of molecular ions. Near the threshold irradiance, there is a precipitous decrease in the probability for producing a desorption event from a given laser pulse, but the number of molecular ions in a given successful event is still large. (ii) On the other hand, the threshold could also be explained by a drastic decline in Author to whom correspondence should be addressed.
the number of ions produced per desorption event, with a consequent decrease in measurement efficiency. In this description, most of the laser pulses continue to produce molecular ions near the irradiance threshold , but the average number of ions produced becomes smaller. If the latter explanation is correct, it seems likely that greater sensitivity could be obtained in the neighborhood of the threshold by using a time-todigital converter (TDC) instead of a transient recorder. The TDC gives a single output pulse for each event detected whose amplitude is larger than a specified value, so it may be optimized for observation of a small number of ions per laser event. It may also be run at a high repetition rate, yielding useful statistics even if the number of events per pulse is low. Here we report the first matrix-assisted LD measurements using single-ion counting. The objective of these experiments was to resolve the question of whether the desorption process is a collective effect. Is it possible to initiate desorption events in which single or very few molecular ions are produced, or is the minimum number of molecular ions per event very large? In addition to improving our understanding of the mechanism, the experiments were motivated by the possibility of improving the spectral quality and increasing the sensitivity if the observation threshold could be lowered. EXPERIMENTAL The schematic arrangement of the experiment is shown in Fig. 1. Here, laser pulses of about 10-2011s in duration from a high-repetition-rate excimer laser (HE460-HR-B) (Lumonics Inc. , Kanata, Ontario, Canada) at 308 nm are incident on the target surface at 45” to the normal. The laser spot size is focused onto an area approximately 50 pm by 80 pm as measured by removal of nitrocellulose from an aluminum target. The power density is controlled in reproducible steps between lo6 and lo7W cm-*, by a series of UV fused silica plates with 8% attenuation per plate. The energy per pulse was measured with a Model 365 power and energy meter (Scientech Inc., Boulder, CO, USA).
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57 Computer
& 'I
1 40 Hz Clock k
-/
Oscilloscope+
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Deflection Plates Figure 1. Schematic diagram of the experimental arrangement. Focusing optics are omitted for clarity. The deflection plates are about 30 cm from the target, and the detector is movable between 40 and 140cm from the target.
Just before entry to the mass spectrometer, the beam passes through a displacement plate mounted on a stepping motor to allow the beam to be rastered across the target. The time-of-flight spectrometer (TOF I, Manit~ba',~), constructed in-house is a simple linear instrument with a single acceleration region (5 cm in this case), a field-free flight path (variable from 40 to 140 cm") and a chevron microchannel plate detector. A variable diameter iris diaphragm is mounted in front of the detector to control the acceptance. Ions are accelerated by applying a potential (typically 14 kV) to the target. The front plate of the detector is set to approximately - 2 kV. The beam can be deflected in 2 perpendicular directions by deflection plates situated 40 cm from the target. The plates are 5 cm square and separated by 3cm. Low-mass ions may be suppressed by pulsing the deflection plates in the field-free region. Amplified signals from the microchannel plates can be observed directly on an analogue oscilloscope, or fed into a 255-stop TDC (model CTN-M2 from Institut de Physique NuclCaire, Orsay, France) connected by a custom interface to an Atari Mega ST computer for data storage and analysis. Bovine insulin was selected as a test compound. Approximately 0.1 g/L (or less) insulin and 10 g/L sinapinic acid were dissolved in 0.1% TFA in water and acetonitrile (1: 4 by volume). Approximately 20 pL of this solution was dropped onto about 3cm2 area of etched silver foil and allowed to dry. The resulting sample thickness was found to give optimum reproducibility for our purposes; it is a few times thinner than that typically used by other Because of the steep dependence of the yield on irradiance, we anticipated that conditions under which single (or a few) ions are produced from a single laser
pulse would be extremely critical (if they existed), and that finding such conditions would be easier starting from high intensity where the experiment was known to work. A TDC (even a multi-stop device) can only record one count in a given time period determined by the dead-time (here 200 ns). In practice this means that no more than one ion of a given mass can .be detected for each laser pulse. If a large number of ions of a given mass strike the detector at the same time, only one pulse is recorded, so no information about the intensity of the signal is obtained. Therefore the procedure outline below was followed in order to get meaningful data with the TDC for irradiances where many ions are produced from a single pulse, as well as for lower irradiance. Starting with an irradiance where many ions of the same mass are produced in a single event (i.e., an irradiance where the detector pulses could be observed on an oscilloscope with the iris fully open), the iris was closed down so that on the average less than one ion per laser pulse passed through the aperture. In that situation, the probability for detection is related to the total ion yield, i.e., it is independent of whether the ions appear in many pulses with only a few ions per pulse, or whether they appear in a smaller number of pulses with more ions per pulse. The average intensity of the signal from a single laser pulse is then represented by the total number of detected ions after many laser shots. With at most one count registered per event for a given mass, a high repetition rate is required to get useful statistics. The excimer laser is capable of running up to 500 Hz but for the present experiment it was not necessary to go beyond 40Hz. A spot on the sample becomes exhausted after about 100 laser shots so it was necessary to move the beam across the target with the displacement plate mentioned above to expose fresh sample to the laser. The beam was rastered over a 5mm square at the centre of the target allowing an array of 40 by 40 spots to be irradiated without overlap. Typically a spectrum could be acquired by irradiating one row of 40 spots. The etched silver targets were selected because the solution spreads evenly over the surface giving reproducibility within about 20% for adjacent rows and within about 50% over the 5mm square examined. When comparisons between many spectra were made, control spectra were run on adjacent rows for normalization. Blocking part of the secondary beam, and rastering the laser is necessary to obtain data in the high irradiance region. In addition, it allows averaging over a large number of laser shots and over a relatively large part of the target, giving considerably better reproducibility than is normally available. Thus the method is useful for the fundamental studies reported here although there may not be any immediate analytical advantages over the standard matrix-assisted LD experiment.
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RESULTS AND DISCUSSION 1. Mass spectra by single-ion counting As a first step toward using a TDC to measure LD spectra, the power density was set just above the threshold for observation of the molecular ion signal on an oscilloscope with the iris open. The iris was then
118 RAPID COMMUNICATIONS IN MASS SPECTROMETRY,VOL. 5, NO. 3, 1991
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closed down to a point where the average number of counts for the insulin molecular ion was less than one per laser pulse. With this irradiance, reasonable statistics were obtained after about 1600 shots on 40 different spots; i.e., 40 shots per spot. The spectrum of bovine insulin obtained in this manner is shown in Fig. 2(a). The deflection plates shown in Fig. 1 were pulsed to suppress low-mass background. As the irradiance is decreased from this level, more spots are required to get useful statistics, or alternatively, the iris diameter can be increased (see below). To illustrate the effect of reducing irradiance, the spectrum in Fig. 2(b) was taken with the same iris diameter but about 1/3 the irradiance used in Fig. 1;the spectrum was accumulated for 8000 laser shots. As reported earlier,7 there is a clear improvement in resolution for the molecular ion, and the Iow-mass background is decreased as the irradiance is decreased. The resolution (mlhm, full-width at half-maximum) obtained at this power density is about 500. This is similar to the resolution reported by Beavis and Chait7 but not significantly better, as might be expected if our irradiance was lower. However, a direct comparison of resolutions is difficult because the conditions are not the same; in particular, the large acceleration region (5 cm) in our spectrometer will reduce resolution significantly if the initial axial velocity spread gives a significant contribution; see below. 2. Irradiance threshold
The results in Fig. 2 indicate an improvement in the spectrum as the irradiance is reduced. However at the lower irradiance the count rate is very low; this may be increased by opening the iris. To determine how far the irradiance could be reduced, the insulin molecular ion yield was measured as a function of irradiance for several different iris diameters. If the threshold for observing molecular ions by laser desorption was instrumental (i.e., related to detection) then it would be expected to decrease for larger iris diameter, since more ions would strike the detector, giving higher probability for detection. The yield increases with the iris diameter as expected (see later discussion and Fig. 6), but to illustrate the variation of yield with irradiance in Fig. 3, we have
"? I 40
[M+H]+ I
I
I
Flight Time (ns) Figure 2. Positive-ion mass spectra of bovine insulin with power densities (a) 6 x lobW cm-', and (b) 2 X lo6W cm-'. For both spec-
tra, light ions were suppressed by pulsing the deflection plates shown in Fig. 1.
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d I
Power Density (W/cm2) Figure 3. Dependence of the insulin molecular-ion yield on irradiance for several different iris diameters: 0 1.5 mm, 3.0 mm,
05.0 mm, A 7.0 mm. The 3 lines indicate scaling with the 4th, 5th and 6th power of the irradiance.
normalized the data for different iris diameters at 3.1 X lo6W cm-2. Different symbols represent different iris diameters. The point on the horizontal axis is an upper limit since no peak was observed there but all the other points represent a detected insulin yield. In each case no insulin signal was observed after the addition of one more attenuator. It is clear from these data that regardless of the iris diameter, the signal disappears at about the same irradiance within about 8% (the attenuation of one plate). Thus the data indicate a true irradiance threshold near 2 X lo6W cm-' for the production of molecular ions. This value of the threshold is only approximate since there is both a temporal and a spatial distribution of deposited energy, neither of which is accurately known; the estimate of 2 X lo6W cm-' was made assuming uniform distributions. It should be noted that our measurements refer only to production of ions, not neutrals. However, the existence of a threshold is predicted by calculations of the total ejecta (ions and neutrals) based on Beer's Law both for thermal ablation" and for a pressure-pulse model. l2 The adiabatic absorption model introduced by Vertes et al. ,I3 also predicts a threshold irradiance for the onset of plasma ionization. Above the threshold irradiance, for at least a small region, the yield increases rapidly, scaling approximately with the 4th to the 6th power of the irradiance. The 3 lines drawn on Fig. 3 indicate the 4th, 5th and 6th power of the irradiance. A 5th power dependence on irradiance has been reported previously for the desorption yield of neutral molecules of tryptophan. l4 3. Collective effects The measurements described above indicate that the total number of ions produced decreases sharply with decreasing irradiance, but do not show how these ions
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are distributed from pulse to pulse. At one extreme, the number of ions desorbed per event might decrease uniformly. At the other extreme, the number of ions per successful desorption event might be constant, with the probability for such an event decreasing with decreasing irradiance. To resolve this question pulseheight distributions were measured at the threshold irradiance for different iris diameters. The pulse-height is a measure of the number of ions that strike the detector. If the number of ions per successful event is large (but the number of successful events is small), then the pulse-height will increase as the iris is opened, since more ions will strike the detector. If the number of ions per event is small but constant, then the pulseheight will remain the same as the iris is opened because, even for an open iris, the average number of detected ions per incident pulse is less than one. The simplest method of determining the pulse-height distributions is to increase the discriminator level. Only pulses higher than the discriminator level are registered in the TDC; for an increment in the level from V to (V-tdV), the decrease in counts corresponds to the number of pulse-heights in that interval. The results are shown in Fig. 4: as the iris diameter is increased, the discriminator level required to reduce the signal becomes larger indicating that the pulse-height from the detector is increasing. For the 25 mm iris, there is no loss of signal until the discriminator level is >200mV. Thus it appears that there is a minimum number of molecular ions in a successful event; i.e., it is a collective effect. Because there is a high ratio of matrix to sample in the substrate, and because the technique is largely insensitive to the protein species15,the collective effect almost certainly depends only on the matrix, i.e., a minimum number of matrix ions must be desorbed in a successful event. Therefore it should be possible to reduce the number of sample ions desorbed per event by reducing the concentration of the sample molecules in the solution. The result of reducing sample concentration by factors of 10 and 100 are shown in Fig. 5. The molecular ion signal drops as expected, as does some of the background, but the signal-to-background ratio becomes progressively worse, suggesting that a significant part of the background has its origin in the matrix. Reduction of the sample concentration by another factor of ten gave no detectable molecular-ion signal
. m
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1.5 mm iris
10.4 0
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.
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200
.
.
.
.
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.
.
400
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.
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.
"
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600
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Discriminator Threshold Figure 4. Insulin molecular-ion yield measured as a function of discriminator level for 3 different iris diameters. The laser irradiance is near the threshold value (2 X lo6W cm-'). The yields are normalized at 7 mV threshold.
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Figure 5. Mass spectra of insulin for 3 different sample concentrations: top, 0.1 g/L; middle 0.01 g/L; bottom, 0.001 g/L. The matrix concentration is 10 g/L in each case and 20 pL of the solution is spread over an area of 1 cm'.
above background. Still, it appears to be possible to acquire useful spectra with significantly reduced sample concentration. In order to estimate the minimum number of molecular ions associated with a successful event, for a given sample concentration, the yield was measured as a function of iris diameter. As long as the average number of ions passing through the aperture for a successful event is much less than one, the measured yield should increase linearly with the area. Once the average number of ions passing through the aperture exceeds the number required for near 100% detection efficiency, every successful event will be detected and the measured yield will saturate. For the lowest sample concentration (0.001 g/L), such a saturation is observed for an iris diameter of about 7mm (Fig. 6(a)). The number of counts measured in a region of low background continues to increase linearly beyond this aperture. Thus there is at least one insulin ion in an area of diameter 7 mm. From measurements of the radial velocity distribution (see below) the ions are distributed over an area of diameter 36 mm for the present field-free path length of 140cm. A rough estimate of the total number of molecular ions that could be detected is then (36/7)' or a few tens of ions. The actual ion yield may be greater since the detection efficiency for a single insulin ion with energy of 16 keV is likely to be considerably less than 100%. The saturation occurs at about 3 mm diameter for the 0.01 g/L sample concentration as shown in Fig. 6(b). This corresponds to several hundred ions per event. For a concentration of 0.1 g/L the yield saturates very close to the minimum iris opening suggesting that 103-104molecular ions are ejected per pulse. It is possible to estimate a charge-to-neutral ratio for the ejected species based on the number of ions ejected from the target, and on the amount of sample removed from the surface. Two assumptions are needed for the estimate to be meaningful: (i) the loss of the molecularion signal due to repeated laser pulses corresponds to the removal of the sample from the irradiated area. Examination of the targets under a microscope after irradiation is consistent with this assumption. (ii) Any
120 RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL 5. NO 3, 1991
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removed sample that does not appear as molecular ions, is ejected as intact neutral molecules; i.e., fragments and clusters (including macroscopic chunks) do not account for a significant amount of the removed material. Identifiable charged fragments have not been observed so this assumption is plausible. There is a signal from negative molecular ions about the same intensity as the positive-ion signal, and a much smaller signal from multiply charged species and from cluster ions. The charge-to-neutral ratio is calculated for 20 pL of 0.1 g/L insulin solution deposited onto 3 cm2 area, or about 1 pmol/mm2, and a laser irradiance near threshold. Under these conditions, the molecular-ion signal disappears after about 100 laser shots as indicated in Fig. 7. Since the beam spot is -0.005 mm2, it appears that 4 x lo7 molecules are removed per laser shot. If the number of molecular ions ejected is -lo4, then there is an excess of neutrals by a factor greater than lo3. A similar charge-to-neutral ratio is observed for particle-induced desorption. l6
-
10 ,001 g/L
1.o
0.0
2.0
3.0
Detector Iris (cm2)
.-
C
.
0.0
.I
.2
.3
Detector Iris (cm2) Figure6. Insulin yield (0)measured as a function of detector-ins diameter. The squares represent background counts measured in a low-background region. (a) Sample concentration 0.001 g/L; (b) sample concentration 0.01 g/L. The matrix concentration was 10 g/L and 2 pL of the solution was deposited on an area of 1cm2.
0
J
0
40
80
120
160
200
24C
Number of shots per spot Figure7. Measured yield for insulin as a function of the number of laser shots per spot. The yield saturates indicating that the signal from a given spot disappears after about 100 shots.
4. Initial velocity distributions Both the radial and axial velocity distributions have an important influence on instrument design and performance. The radial velocity distribution (along with the acceleration voltage), determines the solid angle of ejected ions, and therefore the transmission of the instrument for a given geometry. In the present experiment, the acceptance is particularly important in estimating the total ion yield. The axial velocity distribution and delays in the desorption process or in the extraction of ions affect the resolution and calibration of the spectra. Measurements of the dynamics of the ejection process also contribute to the understanding of the desorption mechanism. Radial uelocity. Radial velocity distributions were measured in 2 perpendicular directions using deflection plates in the field-free region of the spectrometer. Yields of insulin were measured as a function of deflection voltage with an iris diameter of about 1mm for the maximum field-free drift length of 140 cm. The distributions are shown in Fig. 8. From the geometry and the acceleration voltage, the radial velocity may be calculated for ions detected at a given deflection voltage. For the distributions in Fig. 8, the points at half intensity represent ions with a radial velocity u,, corresponding 2.4 eV; the fringing fields of the deflection to mu:/2 plates were taken into account for these calculations. Ions with this radial velocity would strike about 18mm from the centre of the detector if no deflection voltage were present. Thus most of the ions appear within a circle of diameter 36 mm. For a uniform distribution of ions spread over a 36mm diameter circle, a 1mm diameter aperture samples approximately 0.1% of the ions. The width of the radial velocity distribution is comparable to that of insulin ions ejected by keV or MeV particle b~mbardrnent.’~ The radial-velocity distributions for ions from laser desorption appear to be the same for molecular ions and low-mass ions and presumably correspond to emission centred about the target normal. This is also the situation for low-energy bombardment, but is in contrast to MeV bombardment where large molecular ions have radial velocity distributions correlated to the incoming ion direction (although light ions show no such correlation). Axial uelocity. In order to obtain information on the initial axial velocity ( u , ~ )distributions, we measured
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the peak width as a function of path length. This measurement is considerably less direct than the measurement of the radial velocity since there are several possible contributions to the width of the peak; the time-width of the laser pulse, the axial velocity distribution and possibly a spread in the time of emission or extraction. The results for an irradiance of about twice the threshold value are plotted in Fig. 9. The fact that the width increases with flight-path (plotted as flighttime) indicates that an axial velocity spread accounts for at least part of the broadening. The axial velocity spread may be extracted directly from the slope of a line through the data. The 3 lines shown in Fig. 9 are calculated peak widths for 3 axial velocity spreads, indicating that the spread in mu:,/2 is 50 eV for the insulin molecular ion. This result is consistent with that of Beavis and Chait.ls The intercepts of the calculated lines in Fig. 9 correspond to fixed time-spreads which include at least the time-spread during acceleration and the laser-pulse duration. The time-spread during acceleration caused by the axial velocity spread Au,, depends also on the average initial velocity u,,. Because of the large acceleration region (5 cm) in our spectrometer, this spread makes an appreciable contribution to the final peak width. If a very simple picture of ion ejection and extraction is adopted, i.e., if the ions are extracted at the same time from an equipotential plane, then an average initial axial velocity can be extracted from the data. The interce ts of the 3 lines shown in Fig. 9 P 40, 70 and 120 eV, suggesting correspond to muZo/2 that in this description the average initial axial velocity is comparable to the spread in axial velocity.
-
-
n
cn
c. .-
3
2 E
.c.
4
120- Y-deflection 100-80-60-40-200--~~1.~~1~ -600-400-200 0 200 400 600
-600-400-200 0 200 400 600
Deflection Voltage (volts) Figure 8. Yield of the insulin molecular ions measured as a function of deflection voltage. The number of ions detected drops to zero rapidly for a deflection voltage greater than 150 V. Ions detected with this deflection voltage have a radial velocity u, where mu:/2 2.4 eV.
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2401
I100 Figure 9. Insulin peak width measured for different field-free path lengths at constant accelerating voltage. The 3 lines represent the calculated peak width considering only the laser-pulse duration and the initial axial velocity: (a) Uo= 40 eV, AUo= 40 eV; (b) Uo= 70 eV, AUo=50eV, (c) Uo=120eV, AUo=60eV; where Uo=muL/2.
Both the Rockefeller group18 and the Uppsala group" have shown evidence for a spread in emission time as well as in the initial velocity. The data of Fig. 9 can be accounted for without considering such a spread. On the other hand, a more complex process involving time- or spatial-spreads in emission or extraction of the ions cannot be excluded by the data. Such contributions would not affect the slope of the calculated line, so the estimate of the axial velocity spread would be unchanged. However, a different mean initial axial velocity would have to be assumed to fit the data. An axial velocity spread corresponding to 50 eV and a radial velocity spread corresponding to about 3 eV indicates a strongly asymmetric ejection process. The targets used in this experiment were deliberately rendered coarse from acid etching so, on a microscopic scale, there is no well-defined normal direction. The difference in the radial and axial velocity spreads therefore suggests that the process averages over a macroscopic region obscuring the detailed structure. This is consistent with the suggestion that a rapidly expanding macroscopic gas jet is released from the target.6."* 2o An asymmetry between the radial and axial velocity spreads can also be explained by considering the orientation of the acceleration field. If ions are not accelerated by the field until the density of the gas plume is sufficiently low, there will be a spread in the final energy of the ions corresponding to a spread in the distance (2) from the target where unimpeded extraction begins." Such a spread would not affect the radial velocity, but would cause a defect in the axial velocity corresponding to longer flight-times. This explanation is also consistent with observations of the peak shape for increasing irradiance. As shown in Fig. 10, the broadening of the molecular ion peak for insulin with increasing irradiance occurs entirely on the high-time side. The same effect has also been reported by the Uppsala group," but in that case the broadening was much more pronounced for the same increase in irradiance. The difference in the amount of broadening may be understood qualitatively by the fact that the acceleration distance in the present experiment is about 5 times larger than in the Uppsala experiment, reducing the significance of a spatial spread.
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Acknowledgements This work was supported by grants from the US National Institutes of Health (Institute of General Medical Sciences), and from the Natural Sciences and Engineering Research Council of Canada. One of us (F.M.) gratefully ackowledges partial support from the Deutsche Forschungsgemeinschaft. ) I
REFERENCES
v)
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64000 Flight Time (ns)
63000
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Figure 10. Insulin molecular-ion peak for four different irradiances ranging from threshold to about 3 times threshold irradiance. The data are displayed in 32 ns bins smoothed over 7 bins; peak heights are normalized. The peak width increases monotonically with irradiance, all of the broadening taking place on the high-time side.
CONCLUSION We have demonstrated that there is a true irradiance in matrix-assisted laser threshold near lo6W desorption and that the effect is a collective one involving a minimum number of sample ions. This was suggested after the initial discovery of the phenomenon:” but until now the evidence has been indirect. In a typical matrix-assisted laser desorption measurement of bovine insulin, the number of positive molecular ions ejected is lo4. Assuming that the sample is removed when the signal disappears, and that it is ejected as intact molecules, the number of neutrals exceeds the number of ions by a factor of lo3to lo4.The number of molecular ions ejected in one pulse can be reduced by reducing the sample concentration; useful spectra can be obtained with ejection of a few tens of ions. Measurements of initial velocity distributions for bovine insulin indicate a spread in the axial velocity corrsponding to 50 eV. The spread in radial velocity is much smaller; about 2.4eV, thus indicating a strongly asymmetric ejection or extraction mechanism.
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John Wiley & Sons Limited, 1991
Received 12 January 1991; accepted 13 January 1991.
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5. NO. 3. 1991 123
