Mass Spectrometric Analysis of DNA Mixtures: Instrumental Effects

In the present study, instrumental effects on the mass analysis of DNA molecules were investigated quan- titatively using an equimolar DNA mixture. It...
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Anal. Chem. 2003, 75, 5944-5952

Mass Spectrometric Analysis of DNA Mixtures: Instrumental Effects Responsible for Decreased Sensitivity with Increasing Mass Xiaoyu Chen, Michael S. Westphall, and Lloyd M. Smith*

Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706-1396

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has demonstrated great potential to replace gel electrophoresis for DNA sequence analysis. A current limitation in this method is, however, the decreased sensitivity with increasing mass of DNA molecules. In the present study, instrumental effects on the mass analysis of DNA molecules were investigated quantitatively using an equimolar DNA mixture. It is shown that detection efficiency, detector saturation, and ion beam divergence account for the entirety of the observed falloff in signal intensity with increasing mass. Although the present study focused upon the analysis of DNA mixtures, the instrumental effects observed apply equally to other macromolecular mixtures (e.g., proteins, polymers). The development of matrix-assisted laser desorption/ionization (MALDI)1,2 and electrospray ionization3 mass spectrometry in the late 1980s spurred interest in the possibility of using mass spectrometry for DNA sequence analysis. The central technology employed in classical DNA sequence analysis is the electrophoretic separation of single-stranded DNA molecules by size, employed for both the Maxam-Gilbert and Sanger (dideoxy) sequencing methods. Although there are a variety of possible ways in which one might approach the problem of DNA sequencing by mass spectrometry, the most straightforward would be to simply replace this classical electrophoretic size separation with a mass spectrometric alternative. Major advantages of such an approach over conventional electrophoretic separations would potentially include (a) speedspossibly milliseconds to seconds per sample, as opposed to hours for electrophoresis; (b) the elimination of gels from the sequencing process; and (c) a size determination based upon absolute mass, in contrast to electrophoretic mobilities, which are sensitive to modulation by secondary structure and solution conditions. A number of groups, including our own, have actively pursued this goal over the past decade.4-10 Although progress has been * Corresponding author. E-mail: [email protected]. (1) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (4) Parr, G. R.; Fitzgerald, M. C.; Smith, L. M Rapid Commun. Mass Spectrom. 1992, 6, 369-372.

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Figure 1. MALDI-TOF MS analysis of a mixture of seven oligonucleotides showing a decrease in signal intensity with increasing size that is typical for MALDI analysis of oligonucleotide mixtures. The sample was prepared by the dried-droplet method using 3-HPA as matrix, and the sample spot was prepared with ∼5 pmol of each component (prepared from a 5 µM (for each component) equimolar stock solution of the seven oligonucleotides).

made on a number of fronts, the ability of mass spectrometric methods to analyze even fairly simple mixtures of DNA molecules has remained extremely limited. The primary obstacle has been a dramatic decrease in ion signal with increasing mass.5-12 An example of this phenomenon is shown in Figure 1, which shows a typical MALDI-time-of-flight (TOF) spectrum obtained in our laboratory from an equimolar mixture of 7 mixed-sequence oligomers of length ranging from 15 to 51 nucleotides (nt). It is evident from this figure that the ion signal obtained from the higher mass components is much smaller than that obtained from the lower mass components, to the point that the 51-mer component is detected with very poor signal-to-noise ratio. (5) Fitzgerald, M. C.; Zhu, L.; Smith, L. M. Rapid Commun. Mass Spectrom. 1993, 7, 895-897. (6) Wu, K. J.; Shaler, T. A.; Becker, C. H. Anal. Chem. 1994, 66, 1637-1645. (7) Shaler, T. A.; Tan, Y.; Wickham, J. N.; Wu, K. J.; Becker, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 942-947. (8) Mouradian, S.; Rank, D. R.; Smith, L. M. Rapid Commun. Mass Spectrom. 1996, 10, 1475-1478. (9) Nordhoff, E. Trends Anal. Chem. 1996, 15, 240-250. (10) Murray, K. K. J. Mass Spectrom. 1996, 31, 1203-1215. (11) Ebeling, D. D.; Westphall, M. S.; Scalf, M.; Smith, L. M. Anal. Chem. 2000, 72, 5158-5161. (12) Scalf, M.; Westphall, M. S.; Smith, L. M. Anal. Chem. 2000, 72, 52-60. 10.1021/ac030127h CCC: $25.00

© 2003 American Chemical Society Published on Web 09/20/2003

Although there has been one published report showing successful DNA sequence analysis of a sequencing mixture that went out to 100 nt,13 and occasional reports of mass analyses of individual single- or double-stranded DNAs that are much larger,14,15 by and large the mass range limitation illustrated in Figure 1 has restricted most of the work in this area to the analysis of relatively short oligomers. Several reviews on the mass spectrometric analysis of nucleic acids have been published.16-18 There are a number of reasonable hypotheses that one could examine to account for this falloff in signal with increasing mass. These hypotheses fall into two main categories: effects due to characteristics of the mass spectrometric instrumentation employed and effects due to gas-phase or solution-phase chemical behaviors. Possible issues with the instrumentation include the known m/z dependences of electron multiplier and microchannel plate detector responses, detector saturation effects, and m/z dependences of the ion trajectories.19 Possible issues on the chemical side include the known propensity of DNA molecules to solution-20 and gas-phase fragmentation,21 variations in ionization efficiency,22 adduction reactions,22 and suppression effects.23,24 In this paper, it is shown that each of the three instrumentation effects alluded to above introduces a substantial m/z bias into the analysis of DNA mixtures by MALDI-TOF mass spectrometry and that, taken together, these three effects alone are sufficient to account for the entirety of the observed falloff in signal intensity with increasing mass. This result effectively rules out any need to invoke chemical effects to explain the observed falloffs in signal intensity, while also clearly delineating a path forward for the development of new technologies to address these limitations in current instrumentation. EXPERIMENTAL SECTION The work presented here was performed on a modified commercial MALDI-TOF mass spectrometer (Voyager DE-STR, Perspective Biosystems, Framingham, MA). The modifications employed were (a) replacement of the optically coupled single MCP provided, by a Chevron MCP with an anode readout; (b) introduction of an inductive charge detector in front of the MCP. Three separate types of experiments were performed to examine the issues associated with detector sensitivity, detector saturation, and ion beam divergence. Figure 2 shows a schematic diagram of the mass spectrometer system employed. The first grid voltage, V2, and the voltage on the guide wire (not shown in Figure 2), were set at 92% and 0.1% of the accelerating voltage V1, respectively. A microchannel plate detector (MCP) was positioned at the other end of the TOF mass spectrometer and aligned with (13) Monforte, J. A.; Becker, C. H. Nat. Med. 1997, 3, 360-362. (14) Tang, K.; Taranenko, N. I.; Allman, S. L.; Chang, L. Y.; Chen, C. H. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (15) Berkenkamp, S.; Kirpekar, F.; Hillenkamp, F. Science 1998, 281, 260262. (16) Limbach, P. A. Mass Spectrom. Rev. 1996, 15, 297-336. (17) Miketova, P.; Schram, K. H. Mol. Biotechnol. 1997, 8, 249-253. (18) Crain, P. F.; McCloskey, J. A. Curr. Opin. Biotechnol. 1998, 9, 25-34. (19) Schriemer, D. C.; Li, L. Anal. Chem. 1997, 69, 4176-4183. (20) Lindahl, T. Nature 1993, 362, 709-715. (21) Zhu, L.; Parr, G. R.; Fitzgerald, M. C.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1995, 117, 6048-6056. (22) Zenobi, R.; Knochenmuss, R. Mass Spectrom. Rev. 1998, 17, 337-366. (23) Sterner, J. L.; Johnston, M. V.; Nicol, G. R.; Ridge, D. P. J. Mass Spectrom. 2000, 35, 385-391. (24) Wang, M. Z.; Fitzgerald, M. C. Anal. Chem. 2001, 73, 625-631.

Figure 2. Schematic diagram of the experimental apparatus.

the ion beam axis. Except in the study of the ion beam divergence, an inductive charge detector was placed immediately in front of the MCP. This use of a nondestructive inductive charge detector in tandem with an MCP permits calibration of the MCP signal as will be presented in detail below. Responses from both detectors were digitized and processed off-line. All spectra were obtained by averaging 50 laser shots, with a 200-ns delayed extraction time. The design of the inductive charge detector follows that of Fuerstenau and Benner.25 It consists of a copper charge pickup tube, 3.81 cm long and 0.64 cm in inner diameter. This tube is supported with a Teflon insulator inside a stainless steel shielding cylinder. As ions enter the detector, induced charges are formed on the surface of the inner copper tube. The induced signal is converted to a voltage output using a 2N4416 FET coupled to an A250 charge-sensitive preamplifier implanted on a PC250 circuit board (Amptek Inc. Bedford, MA; see Figure 2). The preamplifier is operated in current mode with a feedback resistance of 1 MΩ. The rise and fall times (defined as the 1/e value) of the preamplifier output were observed to be about 40 and 450 ns, respectively. The circuit is shielded by a stainless steel box and placed close to the inductive charge detector inside the vacuum chamber to minimize noise. The output of the preamplifier is directly connected to a Tektronix oscilloscope. The input impedance of the oscilloscope is set to 1 MΩ to match the output impedance of the A250 preamplifier. The microchannel plate detector (3025MA, Burle Electrooptics, Inc., Sturbridge, MA), with a Chevron configuration,26 was placed 4 cm behind the inductive charge detector. Since the MCP is 2.54 cm in diameter, all the ions passing through the 0.64-cmdiameter bore of the inductive charge detector reach the MCP. The MCP was operated in “saturation mode” with 1 kV applied across each of the two plates. Output from the metal anode was ac coupled via a 150-pF capacitor to the oscilloscope, which provided a 50-Ω load. DNA and protein species used in the present work are given in Table 1. All protein samples were obtained from Sigma Chemical Co. (St. Louis, MO) and were dissolved in water to 100 µM. All DNA samples were obtained from Integrated DNA (25) Fuerstenau, S. D.; Benner, W. H. Rapid Commun. Mass Spectrom. 1995, 9, 1528-1538. (26) Wiza, J. L. Nucl. Instrum. Methods 1979, 162, 587-601.

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Table 1. MALDI Samples Used sample

MW

angiotensin substance P neurotensin insulin ovalbumin (dT)5 (dT)25 (dT)100 20-mer oligonucleotide equimolar DNA mixture: 15-mer oligonucleotide 21-mer oligonucleotide 27-mer oligonucleotide 33-mer oligonucleotide 39-mer oligonucleotide 45-mer oligonucleotide 51-mer oligonucleotide

1297 1348 1673 5734 45000 1461 7553 30401 6138 4586 6440 8293 10148 12002 13856 15709

Technologies, Inc. (Coralville, IA). The 20-mer oligodeoxynucleotide (5′-TTT GCC TAA ATG AAA TGT TA-3′), d(T)5, d(T)25, and d(T)100 were obtained HPLC purified and dissolved in water to 100 µM. The seven oligodeoxynucleotides (ranging from 15 to 51 nt in length) used to prepare the DNA mixture were obtained PAGE purified and were further HPLC purified in our laboratory. A 5 µM (for each component) equimolar mixture solution was then made from these purified samples. The concentrations of all DNA solutions were determined based on their absorbance at 260 nm. The 51-mer mixed-base oligonucleotide sequence is as follows: 5′ (TGT AAA ACG ACG GCC AGT GCC AAG CTT GCA TGC CTG CAG GTC GAC TCT AGA) 3′. All other oligonucleotide sequences (15-, 21-, 27-, 33-, 39-, and 45-mer) are identical to the 51-mer sequence, beginning at the 5′ end and ending at the indicated length. Saturated 3-HPA (Aldrich, Milwaukee, WI) in a 1:1:2 mixture of water, acetonitrile, and 0.1 M aqueous diammonium citrate was used as the MALDI matrix. MALDI samples were prepared by combining 1 µL of matrix solution with 1 µL of sample solution on the stainless steel MALDI sample stage, followed by solvent evaporation at atmospheric pressure and temperature. The sample spots were ∼2 mm in diameter. RESULTS AND DISCUSSION Three instrumentation-related factors that might contribute to the observed signal falloff in the MALDI-TOF analysis of DNA mixtures are ion detection efficiency, detector saturation effects, and ion beam divergence. Experimental and theoretical approaches designed to evaluate these factors are presented below, along with the results obtained. A comparison between the signal falloff due to these instrumental effects and the experimental results was made. I. Mass Dependence of Ion Detection Efficiency. MCPs are the most common detectors employed in mass spectrometry of large ions. These detectors operate by means of secondary electrons generated by the initial impact of the ion on the channel surface. The secondary electrons are accelerated down the channel and impact the channel wall, producing additional electrons. Multiple such events lead to an electron cascade. A Chevron MCP has two tandem microchannel plates in close proximity to one another. Secondary electrons exiting a channel 5946 Analytical Chemistry, Vol. 75, No. 21, November 1, 2003

in the front plate enter a number of channels in the back plate and initiate more electron cascades. These electron cascades are subsequently collected at the detector anode, generating an output signal. Thus, the detection efficiency is directly related to the probability of producing one or more secondary electrons from the initial impact of the ion. When operated in “saturation” mode, as is generally the case, the electron cascade depletes the channel of electrons and substantial time (of the order of milliseconds) is required for it to regenerate by current flow from the external electronics. As the millisecond time scale is substantially longer than the duration of the time-of-flight measurement, such channels are effectively disabled for the duration of the measurement and will not respond to subsequent ion or electron collisions until regenerated. Several groups have examined secondary electron emission in ion-surface collisions.27-31 It has been shown that the secondary electron yield decreases significantly as the velocity of the molecular ion decreases. In TOF mass spectrometry, which is the most widely used method for analysis of very large ions, the velocity of the ions decreases as the inverse square root of the mass; as a result, large ions move more slowly and thus have a lower probability of detection. It has also been shown that the chemical composition of the ion can affect its secondary electron emission efficiency.27,28 In 1989, Geno and Macfarlane27 described a universal function to calculate the MCP secondary electron yield for peptides. In subsequent work, Meier and Eberhardt28 measured the gain of an MCP for several atomic and small molecular ions. Recently, by using an energy-sensitive detector, Westmacott et al.29 measured the MCP secondary electron emission efficiency for protein ions. Secondary electron emission has also been studied for protein ions impacting cesium iodide surfaces.30,31 To carefully evaluate the possible role of detector response in the analysis of DNA mixtures, we sought to characterize the MCP response to DNA ions. Although the response to proteins is well documented, little if any work has been done to determine whether response characteristics were similar for DNA ions. We examined the MCP secondary electron emission efficiency for protein and DNA ions ranging in mass from 1300 to 45 000 Da, and with velocities between 5 and 60 km/s. The approach is similar to that utilized by Westmacott et al., who also employed two detectors placed in series, with the difference that in their work the first detector was a superconducting tunnel junction (STJ) detector, whereas the present work utilized a nondestructive inductive charge detector. Spectra of protein and DNA samples were acquired at acceleration voltages of 5, 10, 15, 20, and 25 kV in negative-ion mode for DNA, and in positive-ion mode for proteins. The low-mass gate for the MCP was set at 400 Da to suppress the saturation effect caused by matrix ions. Since both the inductive charge detector and the MCP intercept the same packet of MALDI-generated ions, the ratio of (27) Geno, P. W.; Macfarlane, R. D. Int. J. Mass Spectrom. Ion Processes 1989, 92, 195-210. (28) Meier, R.; Eberhardt, P. Int. J. Mass Spectrom. Ion Processes 1993, 123, 19-27. (29) Westmacott, G.; Frank, M.; Labov, S. E.; Benner, W. H. Rapid Commun. Mass Spectrom. 2000, 14, 1854-1861. (30) Ens, W.; Westmacott, G.; Standing, K. G. Nucl. Instrum. Methods Phys. Res. B 1996, 108, 282-289. (31) Brunelle, A.; Chaurand, P.; Della-Negra, S.; Le Beyec, Y.; Parilis, E. Rapid Commun. Mass Spectrom. 1997, 11, 353-362.

signal intensity between the two detectors can be used to measure the MCP detection efficiency, det. det is defined as the number of detection events per incident ion and can be calculated by

det ) Ne/Nif

(1)

where Ne is the number of events in which one or more initial secondary electrons are ejected from an MCP channel upon impact of an incident ion, Ni is the number of ions traveling through the inductive charge detector and striking the MCP surface, and f, referred to as the open area ratio, is the fraction of the MCP surface that is active. When the MCP is operated in saturation mode, a single initial secondary electron will saturate the channel and produce on average a constant number of secondary electrons, which are subsequently collected by the anode. As a result, Ne can be calculated by

Ne ) Hmcp/G

(2)

where Hmcp is the peak height of the MCP signal observed on an oscilloscope and G is the peak height produced by one initial secondary electron. Theoretical derivations of the signal generated by a point charge moving along the geometrical axis of a conducting ring have been made by Gajewski.32 The maximum value of the voltage drop across the resistance of the measuring device was shown to be proportional to the charge and velocity of the point charge,

Φmax ) ξqv

(3)

Here q is the charge of the ion, v is the velocity of the ion, and ξ is a constant determined by the radius and capacitance of the inductive charge detector, as well as by the input resistance and capacitance of the oscilloscope. The applicability of this equation to the present case of a cylindrical inductive charge detector was confirmed by simulation of the inductive charge detector using the program Maxwell 2D (Ansoft Corp., Pittsburgh, PA) (results not shown). According to (3), the number of ions passing through the inductive charge detector can be calculated by

Ni ) Hc/ξve

(4)

where Hc is the peak height of the induced signal and e is the charge of an electron. Substitution of (2) and (4) into (1) gives

det ) (Hmcp/Hc)vU

(5)

where U ) ξe/Gf. This expression for det permits determination of the detection efficiency from measurement of the signal produced by the inductive charge detector and the MCP in response to a given ion packet. (32) Gajewski, J. B. J. Electrostatics 1984, 15, 81-92.

Ion velocity is an important variable in the present work, since it affects the magnitude of both the inductive charge detector and the MCP signal. Three factors affecting ion velocity were considered: the initial velocities generated in the MALDI process, the velocities imparted to the ions by the applied extraction fields, and the velocities imparted to the ions by small fields at the entrance to the MCP. The first and last of these merit comment, as follows. The initial velocities of ions generated in the MALDI process have been well-studied.33,34 For the same class of analytes, the initial ion velocity in MALDI is independent of the analyte mass and charge and is mainly determined by the matrix employed. Different classes of analytes may yield different initial velocities even for the same matrix. No differences have been found between protein and DNA samples.33 The measured initial velocity also shows a significant dependence on the solvent used in preparation of matrix solution. Gluckman and Karas34 examined the effect of different solvents on initial velocity for the matrix 3-hydroxypicolinic acid (3-HPA). The results range from 444 (water) to 620 m/s (1:1 water/ethanol). In the work of Juhasz et al.,33 3-HPA was dissolved in a 1:1 mixture of water and acetonitrile, and for DNA samples, dibasic ammonium tartrate was added to the matrix solution. The initial ion velocity from this preparation was found to be 537 m/s. Since in the present work, the 3-HPA matrix solution was prepared in a similar manner, we have used this value of 537 m/s for the initial velocity of the DNA and protein ions. At the entrance to an MCP microchannel, an incident ion experiences a small electric field. Positive ions will be decelerated and negative ions accelerated by this field. The magnitude of this effect was measured by Geno and Macfarlane27 for a chevrontype MCP. They found the effective potential to be -50 V. This value is less than 1% of the acceleration potential used in this work and, hence, was neglected here when the ion velocity was calculated. MCP Response to Proteins. Mass spectra of several proteins were acquired with simultaneous detection by both the inductive charge detector and the MCP. Figure 3 shows representative results from both detectors for insulin, acquired using an acceleration voltage of 25 kV. One of the fundamental physical quantities of interest is the secondary electron emission efficiency, e, which is defined as the probability for emitting one or more electrons from the impact of a single molecular ion. Here e g det, and the equal sign holds only if all the detection events are caused by single-ion impacts. If two or more ions enter one channel simultaneously and they were both able to knock out secondary electrons, the observed detection efficiency will be lower than the actual secondary electron emission efficiency, because one single initial secondary electron is enough to saturate the channel. Westmacott et al.29 considered the multiplicity of ions impacting the MCP surface in their study. They found that single-ion impacts accounted for the majority (77%) of the MCP detection events under their experimental conditions. To evaluate the significance of the effect of multiion impacts in our experiments, responses from both detectors for insulin molecular ions (25 keV) were obtained from several locations within several different sample spots. The intrinsic (33) Juhasz, P.; Vestal, M. L.; Martin, S. A. J. Am. Soc. Mass Spectrom. 1997, 8, 209-217. (34) Gluckmann, M.; Karas, M. J. Mass Spectrom. 1999, 34, 467-477.

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Figure 5. Reduced secondary electron yield, γe/m, of the MCP for protein ions. The solid trend line is a fit to the data using the simple power function, γe/m ) AvB, where A ) 10-24(1 and B ) 4.4 ( 0.2. Measurements made by other groups are also plotted for comparison: Geno and Macfarlane data26 (- - -), and Westmacott et al. data28 (- - -).

Figure 3. Mass spectra of insulin (100 µM) acquired with the MCP (a) and the inductive charge detector (b).

angiotensin ion (MW 1297) when accelerated at 25 kV (corresponding to a velocity of ∼61 km/s). This assumption is based upon the results of Geno27 and Westmacott,29 which yield values for e of 1 and 0.99, respectively. U is found to be 1.3 × 10-7 s/m, and this number may then be used to calculate the secondary electron emission efficiency for both protein and DNA ions. The secondary electron yield, γe, is the average number of electrons emitted per molecular ion impact. It can be calculated from the secondary electron emission efficiency,29 e, by

γe ) - ln(1 - e)

Figure 4. Signal intensities from both the MCP detector and the inductive charge detector for insulin molecular ions obtained at a variety of sample spots with an acceleration voltage of 25 kV. The error bars shown denote the rms noise levels for each detector, defined as 1/(21/2) × peak-to-peak baseline noise level.

variability of the MALDI process leads to a corresponding variability in the ion intensities recorded (varying by a factor of ∼20 in these experiments). The ratio of the signal intensity of the MCP to that of the inductive charge detector was examined. It was found that this ratio remained constant across the 20-fold range of signal intensities examined (Figure 4). This shows that the effect of multiion impacts is negligible under the experimental conditions employed, because otherwise, the measured ratio would have decreased with increasing signal intensities (as the probability of multiion impacts increased). Thus we have e ≈ det ) (Hmcp/Hc)vU. In this work, the constant U in (5) was determined by assuming unity secondary electron emission efficiency for an 5948

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(6)

It is well established that the secondary electron yield, γe, depends on both the mass, m, of the incident ion and its velocity, v.27,29-31 The data for γe were plotted as γe/m, the reduced secondary electron yield, versus velocity, to separate the dependence of γe on mass and velocity (Figure 5). The data were fit to a simple power function, γe/m ) AvB. The velocity exponent, B, was found to be 4.4 ( 0.2, and A was found to be 10-24(1. Results from earlier measurements made by other groups are also plotted in Figure 5 for comparison. Westmacott et al.29,35 used a STJ detector to measure the secondary electron emission efficiency of an MCP. The mass and velocity range covered in Westmacott’s measurements is similar to that used here, and their results are highly consistent with those obtained in the present study. The universal peptide function reported by Geno and Macfarlane27 has the form of γe ) 2.58 × 10-7m exp(2.31 × 10-4v). The velocity range used to derive this function is about 15-35 km/s. Our data are consistent with this function within this velocity range as illustrated in Figure 5. Small differences in the magnitudes of the reduced secondary electron yields between earlier results and those reported here are within the experimental error of the present measurements. MCP Response to DNAs. Mass spectra of several deoxyoligonucleotides were acquired with both the inductive charge (35) Westmacott, G.; Zhong, F.; Frank, M.; Friedrich, S.; Labov, S. E.; Benner, W. H. Rapid Commun. Mass Spectrom. 2000, 14, 600-607.

Figure 7. Reduced secondary electron yield, γe/m, of the MCP for DNA ions. The solid trend line is a fit to the data using the simple power function, γe/m ) AvB, where A ) 10-21(1 and B ) 3.8 ( 0.2. The protein results from Figure 5 (dashed line) are also shown for the purpose of comparison.

Figure 6. Mass spectra of d(T)25 (100 µM) obtained with the MCP (a) and the inductive charge detector (b). The first peak at ∼77 µs is the monomer; the second peak at ∼109 µs is the dimer.

detector and the MCP. Figure 6 shows representative results from both detectors for (dT)25, acquired using an acceleration voltage of 25 kV. It is noteworthy that the inductive charge detector’s response to negative DNA ions has a polarity opposite to that obtained from positive protein ions (compare Figures 3b and 6b). This is an important indication that the inductive charge detector response is indeed due to image charge formation, rather than to secondary electron generation. It may be noted that this was not the case for the data presented in ref 36, suggesting that in that case the response was incorrectly attributed to image charge formation. A plot of γe/m versus velocity for DNA ions is shown in Figure 7. A fit to γe/m ) AvB gives 10-21(1 for A and 3.8 ( 0.2 for B. The results from poly(dT) are consistent with those for a 20-mer mixedbase oligonucleotide, indicating that the secondary electron yield for DNA molecules is independent of its base composition. Figure 7 also shows for comparison the fit obtained from the various proteins examined. The closeness of the fit for DNA and proteins shows that there is little difference in secondary electron emission efficiency between these two kinds of molecules. II. Saturation Effects. It is well known in MALDI-TOF studies that the large flux of low molecular weight matrix ions can impair detection sensitivity due to “saturation” effects (see section I). This problem may be addressed by using time-gated detection, in which the voltage applied across the microchannel plate is maintained at a lower level until the ions of interest arrive, at which time the voltage is pulsed to a higher level to “turn on” the detector. (36) Park, M. A.; Callahan, J. H. Rapid Commun. Mass Spectrom. 1994, 8, 317322.

However, even with matrix ions “gated” from the detector, saturation effects can still exist in mixture analysis.37 Ions corresponding to lower molecular weight components in the mixture might suppress the MCP’s sensitivity for ions corresponding to the higher molecular weight components. This has been believed to account for, at least partially, the observation that the molecular weight distributions of polymers determined from mass spectrometry often deviate from those obtained using more classical methods.19,38,39 Accordingly, we undertook a study of these saturation effects in DNA mixture analysis. The detector saturation effects were evaluated experimentally and used to develop a simple quantitative model for the phenomenon. Spectra of the seven-component DNA mixture (ranging from 15 to 51 nt in length, see Experimental Section) were acquired as follows. The trigger pulse of the Voyager mass spectrometer, which is synchronized with the laser pulse, was used to simultaneously trigger the oscilloscope and a pulse generator (100C, Systron Donner, Concord, CA). A delayed output pulse from the pulse generator was then sent to the gate of a high-voltage pulse generator (PVX-4140, Directed Energy, Inc., Fort Collins, CO). The high- and low-voltage levels of this high-voltage pulse generator were set at +2 kV and 0 V, respectively. The output of the high-voltage pulse generator was connected to the back plate of the MCP. The timing and duration of the final high-voltage pulse applied to the MCP were thus controlled by the delay time and duration setting on the 100C pulse generator. The pulses were ∼1 ms in duration, enough to cover the mass range investigated, and delayed by a period of 50-108 µs with respect to the laser pulse, to “gate” the detector at intervals corresponding to the flight times of sequential component ion packets. The saturation effects were eliminated for the first ion packet detected, since the MCP was “off” when the earlier ion packets hit the surface. The response from the inductive charge detector was used to correct the shot-to-shot variations in signal intensity characteristic of (37) Westman, A.; Brinkmalm, G.; Barofsky, D. F. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 79-87. (38) Zhu, H.; Yalcin, T.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 275-281. (39) Rashidzadeh, H.; Guo, B. Anal. Chem. 1998, 70, 131-135.

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Figure 8. Saturation factors for the seven oligodeoxynucleotides in the DNA mixture. The diamonds with error bars are experimental results based on (7). The error bars shown were calculated by propagation of error through (7), where the rms error for each parameter was taken as the 1/(21/2) × peak-to-peak baseline noise level. The triangles are values calculated from (8) using NTG′ ) 0.12.

MALDI, so all the signal intensities obtained after correction could be compared to each other directly. A saturation factor, Si, is defined as

Si )

H1i /Hii

A numerical model was developed to describe the saturation effects associated with the Chevron MCP employed in these studies. A comparison of the manufacturer’s specified gain (4 × 104) for the front and back plates individually, with the overall gain of the combined Chevron MCP (1.5 × 107), indicates that each channel excited in the front plate activates ∼380 channels in the back plate. It is the depletion of the active channels in the back plate which dominates the observed signal saturation in the Chevron MCP. In (7), Hii was obtained with all the exposed channels of the back plate being active, while H1i (i > 1) was obtained with fewer active channels because some of the channels had been depleted by lower molecular weight component ions. Thus, on average, the ratio of H1i to Hii, i.e., Si, equals the ratio of the number of active channels to the total number of channels exposed. The number of channels in the back plate consumed by component j in the mixture is H1j /G′, where G′ is the peak height produced by the electron cascade from one channel. The number of active channels in the back plate available for detection i-1 of component i is therefore NT - ∑j)1 H1j /G′, where NT is the total number of channels exposed in the back plate. Consequently, the saturation factor, Si, can be expressed as

Si ) 1

(7)

for i-1

)1-

∑H j)1

where i stands for different components in the mixture, and 1 e i e 7 in this study with i ) 1 for the 15-mer and i ) 7 for the 51-mer, Hi is the signal intensity measured for component i, and the superscript indicates the component ion packet before which the MCP was turned on; e.g., Hii is the signal intensity for component i with the MCP turned on just before ions of this component hit the detector surface and thus denotes the signal intensity of component i in the absence of the saturation effect. A plot of these measured saturation factors is shown in Figure 8.

i ) 1, 1 j

1 NTG′

for

i>1

(8)

The value of NTG′ was estimated by minimizing the difference between the measured values of Si (see Figure 8) and the values calculated according to (8). This yields a value for NTG′ of 0.12. The saturation factors calculated from (8) using NTG′ ) 0.12 are also shown in Figure 8 for comparison with the experimental results. It may be noted that the assumption here of S1)1, i.e., that there is no significant saturation effect reducing the magni-

Figure 9. Measured signal intensities from the MCP for the seven oligodeoxynucleotides in the DNA mixture. (Note: saturation effects were removed as discussed in section II of the text). The columns on the left in each series are signal intensities with the inductive charge detector in front of the MCP. The columns on the right in each series are signal intensities with the inductive charge detector removed. The data points connected by segments are the percentage of ions detected by the MCP as predicted by the Simion model using an angular distribution of cos2.39θ. The top trace corresponds to simulations with the inductive charge detector in front of the MCP, and the bottom trace corresponds to simulations with the inductive charge detector removed. 5950

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tude of the first peak in the mixture, is validated by the experiments described earlier (see section MCP Response to Proteins) ruling out multiion impacts under the conditions employed here. As shown in Figure 8, the calculated values of the saturation factors are in good agreement with experimental results, validating the theoretical model employed. It is evident from Figure 8 that the saturation effects in the mixture analysis are strongly mass dependent. An ∼4-fold increase in signal intensity would have been observed for the 51-mer in the absence of the detector saturation effect. III. Mass Dependence of Ion Beam Divergence. In MALDITOF, ions are generated with certain initial velocities in both the axial and radial directions. The radial component leads to a cone of ions centered on the central axis of the mass spectrometer. The radius of this cone at the detector end of the instrument can be calculated given the initial angular distribution.40 The signal loss due to ion beam divergence can thus be estimated by comparing the radius of the detector and the radius of the ion beam. However, guide wires are often employed in today’s mass spectrometers, and simple calculations of ion beam radius are not useful since the radial component of ion velocity keeps changing throughout the flight path. In the present study, a Simion model of the mass spectrometer was developed, validated experimentally, and used to estimate the effect of ion beam divergence on the DNA mixture analysis. Simion 7.041 was used to simulate ion trajectories inside the Voyager linear TOF mass spectrometer. In these simulations, 90 molecular ions were created for each of the 7 different mass oligodeoxynucleotides in the mixture. The initial position of each ion was on the center axis and 10 µm from the sample plate. The initial speed of the ions was set to 620 m/s, yielding an average axial velocity of 537 m/s, consistent with the mean axial velocity as measured by Juhasz et al..33 The initial trajectories42-44 were varied by distributing the angle between the ion’s trajectory and the central axis of the spectrometer (located normal to the sample plate surface) in accordance with cos2.39 θi, where θi is the initial desorption angle for ion i (ranging from 0 to 89°).42 All potentials utilized in the simulations were the same as used in actual experiments, including the potentials applied for delayed extraction. At the end of each simulation, the ions’ final positions were recorded together with their initial desorption angles. The percentage of ions (P%) that reached the MCP was calculated by N

∑δ cos θ x

i

P (%) )

i)1

i

× 100

N

(8)

∑ cos θ x

i

i)1

where, N ) 90, δi ) 1 for ions that reached the MCP, δi ) 0 for (40) Axelsson, J.; Scrivener, E.; Haddleton, D. M.; Derrick, P. J. Macromolecules 1996, 29, 8875-8882. (41) Dahl, D. A. SIMION 3D Version 7.0 User’s Manual; Idaho National Engineering and Environmental Laboratory, Idaho Falls, ID, 2000. (42) Spengler, B.; Bokelmann, V. Nucl. Instrum. Methods Phys. Res. B 1993, 82, 379-385. (43) Zhang, W.; Chait, B. T. Int. J. Mass Spectrom. Ion Processes 1997, 160, 259267.

Figure 10. (a) Mass dependence of detection efficiency (calculated using results shown in Figure 7), detector saturation (curve presented in Figure 8 after smoothing), and ion beam divergence, (linear fit to simulation results). The open area ratio of the MCP is a constant (56%). The solid line is the product of these factors and denotes the calculated percentage of ions detected by the MCP. (b) Comparison between the signal intensity obtained from MALDI-TOF analysis of DNA mixtures (columns) and calculated percentage of ions detected by the MCP (solid line). The MALDI-TOF data were averaged over 10 measurements, and the error bars denote the noise level of the MCP response.

ions that did not, and x ) 2.39. The validity of the Simion model was tested by comparing the predicted and measured MCP responses with and without the inductive charge detector (serving in this case as an aperture passing the ions close to the spectrometer axis, but blocking ions further from the axis) in front of the MCP. The MCP response for molecular ions of each component in the DNA mixture was obtained by averaging over 10 measurements, with the results shown in Figure 9. It is clear that there is good agreement between the predicted and measured ion signals, indicating that the Simion model provided an accurate description of the ion trajectories. IV. Combined Instrumental Effects upon DNA Mixture Analysis. The work presented in sections I-III above provides direct quantitative measures of the m/z dependence of detection efficiency, detector saturation, and beam divergence upon measured MCP response in the MALDI analysis of DNA mixtures. These results are plotted in Figure 10a. Another detector-related factor, the open area ratio (f) of the MCP detector, was also included in Figure 10a. Since f is a constant (56%, according to (44) Ayala, E.; Costa Vera, C.; Hakansson, P. Rapid Commun. Mass Spectrom. 1999, 13, 792-797.

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Figure 11. Contribution of instrumental effects upon observed signal falloff in DNA mixture analysis. The percentage of ion loss caused by these instrumental effects are calculated in the order of ion beam divergence, open area ratio of the MCP, detection efficiency, and detector saturation as discussed in section IV of the text.

the manufacturer’s quote, for the MCP employed in this work) for a given MCP, this factor does not contribute to the decreased sensitivity with increasing mass. Figure 10b shows a comparison of the measured MCP response to the DNA mixture and the predicted response based upon these instrumental factors, and assuming equimolar ion production in the MALDI source for each mixture component. The agreement is excellent, showing that these effects together are sufficient to account for the observed signal intensity decrease. It is also evident from Figure 10 that only a small portion of the ions are detected for each component. It is useful to consider the effects of the instrumental factors upon ion loss in the following order: ion beam divergence, open area ratio, detection efficiency, and detector saturation. Ion beam divergence is the first source of ion loss, since some ions do not reach the detector. The open area ratio, f, determines the next source of ion loss, since some ions hit the detector-inactive area. The MCP detection efficiency comes into play next, as not all ions impacting on channel walls initiate an electron cascade. The final source of ion loss is due to detector saturation, as secondary electrons are not generated in front or back plate channels that have already been depleted. The contribution of each of these

instrumental factors to the total percentage loss of ions is illustrated in Figure 11. This analysis clearly reveals that, in order to extend the mass range of DNA mixture analysis to larger molecules (for example, conventional DNA sequencing by gel electrophoresis is able to resolve DNA molecules differing in length by only 1 nucleotide out of 100045), a fruitful direction to explore will be the development of more sensitive detectors and more efficient ion optics. Although the present work has focused upon the analysis of DNA mixtures, the mass biases delineated here are the result of general instrumental effects and thus will apply equally to any macromolecular mixture. For example, a recent publication showed a dramatic decrease in signal intensity with increasing mass for equimolar mixtures of both proteins and polymers,46 underscoring the generality of this phenomenon.

(45) Salas-Solano, O.; Carrilho, E.; Kotler, L.; Miller, A. W.; Goetzinger, W.; Sosic, Z.; Karger, B. L. Anal. Chem. 1998, 70, 3996-4003. (46) Twerenbold, D.; Gerber, D.; Gritti, D.; Gonin, Y.; Netuschill, A.; Rossel, F.; Schenker, D.; Vuilleumier, J. Proteomics 2001, 1, 66-69.

Received for review March 27, 2003. Accepted July 9, 2003.

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ACKNOWLEDGMENT We thank Dr. Travis Berggren and Dr. Brian Frey for their critical review of this paper. This work was supported by NIH Grant R01HG01808.

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