RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6,209-213 (1992)
Matrix-assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids T. Huth-Fehre,' J. N. Gosine,* K. J. Wu and C. H. Becker* Molecular Physics Laboratory, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
SPONSOR REFEREE: Professor Peter Williams, Department of Chemistry, Arizona State University, Tempe, AZ, USA
Ferulic acid, sinapinic acid and 2,s-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T),z and a mixture of oligonucleotides pd(T)lz through pd(T),8using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.
Since its introduction in 19881,2matrix-assisted laser desorption (MALD) mass spectrometry has found widespread use in the analysis of biopolymers, especially polypeptides and proteins (for a brief review see the article by Widmer et d 3 )Most . classes of biologically relevant materials have been successfully accessed by this method, but only relatively few results have been reported for the mass spectrometry on unprotected ~ingle-stranded~-~ or double-stranded4 oligonucleotides. In contrast to the situation for the desorption of polypeptides and proteins, where molecules in the mass range of 10000-100000Da are analysed r ~ u t i n e l y ,MALD ~.~ of single-stranded oligonucleotides seems to be more difficult with lower mass limits to date. None of the publications cited above presents data for molecules containing more than 10 bases, or of molecular weight roughly 3000 Da. If this mass limit could be brought up to -100kDa with a mass resolution of -500, mass spectrometry would be a fast alternative for directly replacing gel electrophoresis for DNA sequencing. This paper presents some initial results investigating the MALD approach for single-stranded oligonucleotides with regard to mass resolution and mass-range limitations. In this study we compare the performance of three matrix materials, commonly used for protein analysis, under various optimized desorption conditions for oligodeoxythymidylic acids which were chosen as a test material. All three substances, ferulic acid,' sinapinic acid (SA),9 and 2,5-dihydroxybenzoic acid (DHBA)" are aromatic organic acids with strong absorption at 337 nm (nitrogen laser emission), a wavelength chosen because it avoids resonant absorption by the DNA oligomers.
EXPERIMENTAL Desorption and simultaneous ionization of the samples were caused by illumination with 5 ns pulses of nitrogen laser radiation at 337 nm using a model EMG 500 laser
' Present address: Department of Radiation Sciences, Uppsala University, 751 21 Uppsala, Sweden. Present address: Department of Chemistry, University of California at Davis, Davis, CA 95616, USA. Author to whom correspondence should be addressed 095 1-41 98/92/030209-05 $05.OO
01992 by John Wiley & Sons, Ltd.
(Lambda Physik, Gottingen, Germany). The desorbing laser beam was spatially filtered and subsequently focused onto the sample with a 300mm focal length quartz lens to a spot size of approximately 300X 500pm-a value derived from burn marks on heat sensitive paper. By varying the discharge voltage of the laser and by inserting quartz glass windows into the beam, the energy density per pulse delivered to the sample was varied between 10 and 100 mJ/cm2. The ions produced in desorption were extracted perpendicular to the sample surface into the reflecting time-of-flight mass spectrometer (TOF-MS) by biasing the sample with a voltage of - 2.5 keV and keeping the drift and extraction potentials at ground potential. This resulted in an extraction field of approximately 600 V/mm. An einzel lens about 5 c m from the sample focused the ions. This TOF-MS has a two-stage electrostatic reflector, an apparent drift path of 1.96 m and a dual microchannel plate detector. The front surface of the detector was kept at ground potential when detecting negative ions, resulting in an impact energy of 2.5 keV. To avoid detector saturation for the oligonucleotides, due to the high abundance of ionized matrix molecules, the matrix ions were deflected from the detector by a pulsed electrical field of 200V/cm. The signal output of the microchannel plates was amplified and then digitized using a model 2101ASA digitizer (DSP Technology, Fremont, CA, USA) with a time resolution of 20 ns/channel and typically averaged over 50 to 100 laser pulses at a given sample location. To improve the signal-to-noise ratio and clarity in the peak structure for some of the spectra, the data were then smoothed by a simple arithmetic average over several adjacent channels. The loss in mass resolution caused by this smoothing procedure was negligible compared to the width of the unresolved isotopic distributions. However, the raw data are always shown for comparison when this smoothing is used. Mass calibration for direct ionization by desorption was performed by analyzing a mixture of carbon fullerenes; using the isotopically resolved mass peak l2C,, (mlz 1008) as the highest calibration mass, we estimate the absolute mass accuracy at mlz 4000 to be better than k 3 u. Ferulic acid (4-hydroxy-3-methoxycinnamic acid, Chemical Abstract Service (CAS) # [1135-24-61), sinaReceived 3 January 1992 Accepted 5 January I992
MALD MS OF OLIGODEOXYTHYMIDYLIC ACIDS
210 450
M t 134Da
I
LYV
3000
3500
4000
4500
m/’ Figure 1. Time-of-flight mass spectrum of negative ions from pd(T),, laser-desorbed from a ferulic acid matrix by 337 nm light at 32mJ/cm2, resulting from summing over 100 laser pulses (raw data). The molar ratio of ferulic acid to oligomer was 2000:l.
pinic acid (3,5-dimethoxy-4-hydroxycinnamicacid, CAS # [530-59-61), and DHBA (2,5-dihydroxybenzoic acid CAS # [490-79-91, also called gentisic acid) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). All three substances were used without further purification and dissolved to concentrations of 0.1 mol/L. For ferulic acid, 80% methanol in water was used as a solvent, while the other two matrix substances were dissolved in a mixture of 50% acetonitrile, 45% water and 5% ethanol. These solvents were chosen to achieve the most homogeneous crystallization patterns in the air-dried final samples. The oligonucleotides used, pure pd(T),, and a mixture of pd(T)12-18,were obtained from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ, USA) as sodium salts and dissolved in deionized water to concentrations of 40 pmol/L without further purification. By mixing the two solutions in a vial, molar ratios of matrix to analyte between 2000:l and 10 000 :1 were produced. Drops containing 2 0 p L of these final mixtures were applied to an aluminium probe tip, spread to an area of about 0.5 cm2 and dried in a gentle stream of warm air prior to insertion into the vacuum system. This preparation resulted in 30 to 400 pmol of analyte deposited on the probe tip; however, only a tiny fraction of this material was consumed. RESULTS AND DISCUSSION Figure 1 shows the best results we could obtain in terms of mass resolution and minimum adduct formation for the laser-desorption of pd(T),, with ferulic acid as matrix substance. The peak is almost 100 Da wide and is centered 134 Da above the calculated monoisotopic mass of 3666 Da for the analyte with complete substitution of hydrogen for the sodiums. The optimum compromise between minimizing dimer formation of the analyte and poor signal-to-noise ratio was found at a molar ratio of matrix to analyte of 2000:l. However, dimer signals were never very intense or well defined in terms of mass resolution. An optimum energy density for desorption of 32mJ/cm2 was found. Varying this energy density up or down by 20% already produced strand scission, visible in the appearance of a similar broad peak -300 Da lighter than the primary feature shown in Fig. 1. The breadth of the mass peak in Fig. 1
as well as its shift to higher masses of 134Da may be due to a combination of several adduct and fragmentation peaks, unresolvable in our mass spectrometer because of a high kinetic energy spread and/or metastable decay of the ions, indicative of a relatively energetic and/or long-lasting desorption process (that is, ions created over a substantial distance from the surface). The relative contributions of these factors is not clear at this time. Using sinapinic acid as a matrix for P ~ ( T ) ~gave , , the results in Fig. 2, which show a little more detail than those in Fig. 1. Adduction of a whole matrix molecule forms the prominent peak in this mass spectrum, labelled ‘M+SA’, but the width of this peak is still almost 50Da. The unlabelled peak at slightly lower masses than the M + SA labelled peak (about 44 Da less than the M + SA peak) appears to correspond to adduction of decarboxylated sinapinic acid. Again, the optimum molar ratio was 2000:1, and precise adjustment of the desorption energy, to 3 8 + 5 mJ/cm*, was necessary to minimize fragmentation. At lower values of laser energy density, adduct formation was found to increase. Labels such as ‘M+2SA’ in Fig. 2 and other figures are drawn to the calculated position for the mass and not to any particular feature. Figure 3 shows a mass spectrum of pd(T),, desorbed from a DHBA matrix. Optimum mass resolution and signal-to-noise ratio in the spectrum in this case was achieved with a matrix :analyte molar ratio of 6000:1, and a laser energy density of 62mJ/cm2. Most of the peaks can be identified as adducts and no distinct peak is visible below 3660 Da, thus identifying the first peak
300 280
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260
w
4
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(b)
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280 > k cn
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260
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4
240
U W
220
LVV
3000
4000
3500
4500
m/z Figure 2. TOF mass spectrum of negative ions from pd(T),, laserdesorbed from a sinapinic acid (SA) matrix by 337nm light at 38 mJ/cm2resulting from summing over 100 laser pulses, showing (a) the raw data and (b) the result of 5 point smoothing of the spectrum. The molar ratio of sinapinic acid to oligomer was 2000:l.
MALD MS O F OLIGODEOXYTHYMIDYLIC ACIDS
211
2500
2400
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2300
za
2200
z
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2100
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4000
5000
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7000
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2300
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2300
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2000 3000
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4
4000
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2100
Figure3. TOF mass spectrum of negative ions from pd(T),, laserdesorbed from a DHBA matrix by 337 nm light at 62 mJ/cm2 resulting from summing over 200 laser pulses, showing (a) the raw data and (b) the result of 5 point smoothing of the spectrum. The molar ratio of DHBA to oligomer was 6000:l.
as the deprotonated unfragmented negative ion of the analyte. The substitution of a proton by a sodium ion can be observed, probably occurring in a phosphate group in the backbone of the 01igomer.~ The unidentified adduct feature around 3730 Da, the matrix adduct peak, and the broad unresolved background bump between 3100 and 4700 Da were found to be much smaller in a few 'good' spots on a similarly prepared sample, as depicted in Fig. 4. Here the analyte with no sodium attachment and the single-sodiumexchanged molecule dominate the spectrum, most 800
[M-2HtNa]I
[M-H1-ll
qnn L""
3000
3500
2200
4000
4500
m/z
Figure4. TOF mass spectrum of negative ions from pd(T),* laserdesorbed out of a DHBA matrix by 337nm light at 62mJ/cm2, resulting from summing over 200 laser pulses (raw data). A similarly prepared sample and the same desorption conditions as in Fig. 3 were used.
2000
'
3000
4000
5000
6000
I 7000
m/z
Figure5 TOF mass spectrum of negative ions from a mixture of pd(T),, through pd(T)IBlaser-desorbed from (a) a DHBA matrix resulting from summing over 50 laser pulses, and (b) a sinapinic acid matrix resulting from over lo00 laser pulses.
likely as deprotonated species. The accuracy of the absolute mass scale of k 3 Da does not allow us to distinguish between deprotonation and electron adduction in this work, but deprotonation is already known and as the dominant mechanism for negative charging of oligonucleotides. However, the mass resolution and relative accuracy of the mass scale are high enough to show a mass difference of 22 between the main peaks instead of 23, hence proving the exchange of a proton by a sodium ion. Interestingly, only one exchange per molecule is detectable. Presuming equal probabilities for all 12 phosphate groups to undergo an exchange, a third main peak about half as intense as the other two showing two sodium atoms on the same molecule should be visible. Its absence suggests the existence of a preferred binding site of the sodium, which might be found in the phosphate at the 5' end of the chain. It is not clear why such extensive protonation occurs after starting with the sodium salt of the DNA oligomer, even if there is a preferred binding site for the sodium. This lack of sodium attachment seems fortuitous for the laserdesorption mass spectrometry, especially when compared with the case for electrospray ionization mass spectrometry where significant efforts to remove the sodium ions is required for superior mass spectra." In order to test the analysis of oligonucleotide mixtures , a mixture of 7 different oligothyminosines (pd(T),Z-pd(T)lR)was desorbed from a DHBA matrix at a total molar ratio of 10 000 :1 and a 337 nm laser
MALD MS O F OLIGODEOXYTHYMIDYLIC ACIDS
212
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110
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4000
.
5000
6000
7000
m/z Figure6. Smoothed versions of Fig. 5(a) and (b), with 15 point smoothing for part (a) and 5 point smoothing for part (b).
energy density of 52mJ/cm2. The results (Fig. 5(a)) show 7 equally spaced peaks at the expected masses, rising by a factor of 2 to 4 above a broad background hump probably consisting of unresolved fragment and adduct peaks with substantial metastability. Only the first peak (pd(T),J shows no fragmentation, which appears to increase with increasing length of the molecule. For comparison, the same mixture of pd(T),, to pd(T),* was desorbed from a sinapinic acid matrix at a molar ratio of 1OOOO:l shown in Fig. 5(b). All seven peaks are distinguishable again, but the unresolved background is more substantial in this case. No fine structure is revealed although the data are averaged over 1000 laser pulses, instead of 50 in Fig. 5(a). Figure 6(a) and (b) show the data of Fig. 5 with some smoothing. In general, the ferulic acid and sinapinic acid matrices show not only greater propensity for adduct formation with the oligomers, but also considerable peak broadening. This broadening may be from one or more of several sources. It may be due to ion formation over as much as several hundred micrometers above the sample surface as a result of ion/molecule reactions from a high density expansion, or possibly a large matrix-analyte cluster emitting molecules over a similarly significant distance above the surface. Both of these situations would lead to very significant kinetic energy spreads. Metastable decomposition in the extraction region of the mass spectrometer similarly will result in significant peak broadening.” In contrast, the kinetic energy spread expected from the vapor expansion extrapolated from a previous measurement of desorbed neutral gramicidin S from ferulic acid matrixL3would suggest only a few eV kinetic energy
spread in this mass range which should be compensated by the reflector. Local charging on relatively thick insulating samples could conceivably also produce broadening of the mass spectrum due to variations in the electric potential at the point(s) of ion creation; however we have no reason to believe that this is responsible for the observed differences between the different matrices or on ,different locations on a given matrix because of the generally consistent thicknesses of the samples. Another obvious difficulty encountered especially with the DHBA matrix for these DNA oligomers is the nonuniformity of sample. An improved mass spectrum (Fig. 4 vs Fig. 3) was obtained for this system simply by finding an optimum location on the sample under the desorbing laser beam even though considerable care had gone into obtaining uniform crystallization. In fact, cleavage of a relatively large single crystal of the DHBA-oligomer system with a razor blade and subsequent analysis just resulted in mass spectra like the one shown in Fig. 3. At this time, we do not know what exactly is responsible for producing an optimum vs nonoptimum location on the sample in terms of different crystallization. Furthermore, finding these optimum locations currently is simply a matter of scanning the sample position under the laser beam with an x-y manipulator and observing the mass spectrum in realtime on an oscilloscope until an apparent ‘good spot’ is found; this obviously is an undesirable approach in the long run. CONCLUSION Three aromatic organic acids have been examined as matrices for matrix-assisted UV laser desorptionl ionization of synthetic thyminosine oligomers pd(T)12 to pd(T),, used as test species for single-stranded DNA. The DHBA matrix proved to produce the best mass spectra, allowing resolution of salt adducts and very clear identification of the components of a mixture of oligomers from a 12-mer to an l&mer. Relatively little sodium adduction is observed. The ferulic acid and sinapinic acid matrices suffered from significant adduct formation and peak broadening. These mass spectra show that the matrix-assisted laser desorption method holds significant promise as a future technology to directly replace gel electrophoresis in DNA sequencing. However, improvement will be required in extending the mass range by at least an order of magnitude which currently appears to be limited in these instances due to fragmentation. Major improvements in the matrix-assisted laser desorption mass spectrometry method might lie in finding alternative matrix substances and/or conditions in order to’ produce even more favorable ionization conditions. Acknowledgements The authors thank Dr C. Green for helpful discussions. Financial support from NIH (Grant No. HG00174-02) and Deutsche Forschungsgemeinschaft (fellowship for THF) is gratefully acknowledged.
REFERENCES 1. M. Karas and F. Hillenkamp, Anal. Chem. 60, 2299 (1988). 2. K. Tanaka, H. Waki, Y. Ido, S. Akita, Y. Yoshida and T. Yoshida, Rapid Commun. Mass Spectrom. 2, 151 (1988).
MALD MS OF OLIGODEOXYTHYMIDYLIC ACIDS 3. H. M. Widmer, K. 0. Bornsen and M. Schar, Chimia 44,417 (1990). 4. R. W. Nelson, R. M. Thomas and P. Williams, Rapid Commun. Mass Spectrom. 4, 348 (1990). 5 . K. 0. Bornsen, M. Schar and H . M. Widmer, Chimia 44,412 (1990). 6 . B. Spengler, Y. Pan, R. J. Cotter and L.-S. Kan, Rapid Commun. Mass Spectrom. 4, 99 (1990). 7. R. Hettich and M. Buchanan, J. A m . SOC.Mass Spectrom. 2,402 (1991). 8. R. C. Beavis and B. T. Chait, Proc. Nut1 Acad. Sci. USA 87,
213
6873 (1990). 9. R. C. Beavis and B. T . Chait, Rapid Commun. Mass Spectrom. 3, 432 (1989). 10. B. Stahl, M. Streup, M. Karas and F. Hillenkamp, Anal. Chem. 63,1463 (1991). 11. J . T. Stults and J . C. Marsters, Rapid Commun. Mass Spectrom. 5 , 359 (1991). 12. B . Spengler, D. Kirsch and R. Kaufmann, Rapid Commun. Muss Spectrom. 5 , 198 (1991). 13. T. Huth-Fehre and C. H . Becker, Rapid Commun. Mass Spectrom. 5 , 378 (1991).
Matrix-assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids T. Huth-Fehre,' J. N. Gosine,* K. J. Wu and C. H. Becker* Molecular Physics Laboratory, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025, USA
SPONSOR REFEREE: Professor Peter Williams, Department of Chemistry, Arizona State University, Tempe, AZ, USA
Ferulic acid, sinapinic acid and 2,s-dihydroxybenzoic acid (DHBA) have been tested as matrix materials for matrix-assisted laser desorption of the pure oligonucleotide pd(T),z and a mixture of oligonucleotides pd(T)lz through pd(T),8using pulsed 337 nm radiation combined with reflecting time-of-flight mass spectrometry. The three matrix materials are compared with respect to obtainable mass resolution, degree of fragmentation, and adduct formation for these oligonucleotides. DHBA was found to produce the least fragmentation and adduct formation, as well as the highest mass resolution.
Since its introduction in 19881,2matrix-assisted laser desorption (MALD) mass spectrometry has found widespread use in the analysis of biopolymers, especially polypeptides and proteins (for a brief review see the article by Widmer et d 3 )Most . classes of biologically relevant materials have been successfully accessed by this method, but only relatively few results have been reported for the mass spectrometry on unprotected ~ingle-stranded~-~ or double-stranded4 oligonucleotides. In contrast to the situation for the desorption of polypeptides and proteins, where molecules in the mass range of 10000-100000Da are analysed r ~ u t i n e l y ,MALD ~.~ of single-stranded oligonucleotides seems to be more difficult with lower mass limits to date. None of the publications cited above presents data for molecules containing more than 10 bases, or of molecular weight roughly 3000 Da. If this mass limit could be brought up to -100kDa with a mass resolution of -500, mass spectrometry would be a fast alternative for directly replacing gel electrophoresis for DNA sequencing. This paper presents some initial results investigating the MALD approach for single-stranded oligonucleotides with regard to mass resolution and mass-range limitations. In this study we compare the performance of three matrix materials, commonly used for protein analysis, under various optimized desorption conditions for oligodeoxythymidylic acids which were chosen as a test material. All three substances, ferulic acid,' sinapinic acid (SA),9 and 2,5-dihydroxybenzoic acid (DHBA)" are aromatic organic acids with strong absorption at 337 nm (nitrogen laser emission), a wavelength chosen because it avoids resonant absorption by the DNA oligomers.
EXPERIMENTAL Desorption and simultaneous ionization of the samples were caused by illumination with 5 ns pulses of nitrogen laser radiation at 337 nm using a model EMG 500 laser
' Present address: Department of Radiation Sciences, Uppsala University, 751 21 Uppsala, Sweden. Present address: Department of Chemistry, University of California at Davis, Davis, CA 95616, USA. Author to whom correspondence should be addressed 095 1-41 98/92/030209-05 $05.OO
01992 by John Wiley & Sons, Ltd.
(Lambda Physik, Gottingen, Germany). The desorbing laser beam was spatially filtered and subsequently focused onto the sample with a 300mm focal length quartz lens to a spot size of approximately 300X 500pm-a value derived from burn marks on heat sensitive paper. By varying the discharge voltage of the laser and by inserting quartz glass windows into the beam, the energy density per pulse delivered to the sample was varied between 10 and 100 mJ/cm2. The ions produced in desorption were extracted perpendicular to the sample surface into the reflecting time-of-flight mass spectrometer (TOF-MS) by biasing the sample with a voltage of - 2.5 keV and keeping the drift and extraction potentials at ground potential. This resulted in an extraction field of approximately 600 V/mm. An einzel lens about 5 c m from the sample focused the ions. This TOF-MS has a two-stage electrostatic reflector, an apparent drift path of 1.96 m and a dual microchannel plate detector. The front surface of the detector was kept at ground potential when detecting negative ions, resulting in an impact energy of 2.5 keV. To avoid detector saturation for the oligonucleotides, due to the high abundance of ionized matrix molecules, the matrix ions were deflected from the detector by a pulsed electrical field of 200V/cm. The signal output of the microchannel plates was amplified and then digitized using a model 2101ASA digitizer (DSP Technology, Fremont, CA, USA) with a time resolution of 20 ns/channel and typically averaged over 50 to 100 laser pulses at a given sample location. To improve the signal-to-noise ratio and clarity in the peak structure for some of the spectra, the data were then smoothed by a simple arithmetic average over several adjacent channels. The loss in mass resolution caused by this smoothing procedure was negligible compared to the width of the unresolved isotopic distributions. However, the raw data are always shown for comparison when this smoothing is used. Mass calibration for direct ionization by desorption was performed by analyzing a mixture of carbon fullerenes; using the isotopically resolved mass peak l2C,, (mlz 1008) as the highest calibration mass, we estimate the absolute mass accuracy at mlz 4000 to be better than k 3 u. Ferulic acid (4-hydroxy-3-methoxycinnamic acid, Chemical Abstract Service (CAS) # [1135-24-61), sinaReceived 3 January 1992 Accepted 5 January I992
MALD MS OF OLIGODEOXYTHYMIDYLIC ACIDS
210 450
M t 134Da
I
LYV
3000
3500
4000
4500
m/’ Figure 1. Time-of-flight mass spectrum of negative ions from pd(T),, laser-desorbed from a ferulic acid matrix by 337 nm light at 32mJ/cm2, resulting from summing over 100 laser pulses (raw data). The molar ratio of ferulic acid to oligomer was 2000:l.
pinic acid (3,5-dimethoxy-4-hydroxycinnamicacid, CAS # [530-59-61), and DHBA (2,5-dihydroxybenzoic acid CAS # [490-79-91, also called gentisic acid) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). All three substances were used without further purification and dissolved to concentrations of 0.1 mol/L. For ferulic acid, 80% methanol in water was used as a solvent, while the other two matrix substances were dissolved in a mixture of 50% acetonitrile, 45% water and 5% ethanol. These solvents were chosen to achieve the most homogeneous crystallization patterns in the air-dried final samples. The oligonucleotides used, pure pd(T),, and a mixture of pd(T)12-18,were obtained from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ, USA) as sodium salts and dissolved in deionized water to concentrations of 40 pmol/L without further purification. By mixing the two solutions in a vial, molar ratios of matrix to analyte between 2000:l and 10 000 :1 were produced. Drops containing 2 0 p L of these final mixtures were applied to an aluminium probe tip, spread to an area of about 0.5 cm2 and dried in a gentle stream of warm air prior to insertion into the vacuum system. This preparation resulted in 30 to 400 pmol of analyte deposited on the probe tip; however, only a tiny fraction of this material was consumed. RESULTS AND DISCUSSION Figure 1 shows the best results we could obtain in terms of mass resolution and minimum adduct formation for the laser-desorption of pd(T),, with ferulic acid as matrix substance. The peak is almost 100 Da wide and is centered 134 Da above the calculated monoisotopic mass of 3666 Da for the analyte with complete substitution of hydrogen for the sodiums. The optimum compromise between minimizing dimer formation of the analyte and poor signal-to-noise ratio was found at a molar ratio of matrix to analyte of 2000:l. However, dimer signals were never very intense or well defined in terms of mass resolution. An optimum energy density for desorption of 32mJ/cm2 was found. Varying this energy density up or down by 20% already produced strand scission, visible in the appearance of a similar broad peak -300 Da lighter than the primary feature shown in Fig. 1. The breadth of the mass peak in Fig. 1
as well as its shift to higher masses of 134Da may be due to a combination of several adduct and fragmentation peaks, unresolvable in our mass spectrometer because of a high kinetic energy spread and/or metastable decay of the ions, indicative of a relatively energetic and/or long-lasting desorption process (that is, ions created over a substantial distance from the surface). The relative contributions of these factors is not clear at this time. Using sinapinic acid as a matrix for P ~ ( T ) ~gave , , the results in Fig. 2, which show a little more detail than those in Fig. 1. Adduction of a whole matrix molecule forms the prominent peak in this mass spectrum, labelled ‘M+SA’, but the width of this peak is still almost 50Da. The unlabelled peak at slightly lower masses than the M + SA labelled peak (about 44 Da less than the M + SA peak) appears to correspond to adduction of decarboxylated sinapinic acid. Again, the optimum molar ratio was 2000:1, and precise adjustment of the desorption energy, to 3 8 + 5 mJ/cm*, was necessary to minimize fragmentation. At lower values of laser energy density, adduct formation was found to increase. Labels such as ‘M+2SA’ in Fig. 2 and other figures are drawn to the calculated position for the mass and not to any particular feature. Figure 3 shows a mass spectrum of pd(T),, desorbed from a DHBA matrix. Optimum mass resolution and signal-to-noise ratio in the spectrum in this case was achieved with a matrix :analyte molar ratio of 6000:1, and a laser energy density of 62mJ/cm2. Most of the peaks can be identified as adducts and no distinct peak is visible below 3660 Da, thus identifying the first peak
300 280
> t (0
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260
w
4
240
LT W
220
(b)
MtSA
280 > k cn
5 cz
N
SA Mt3SA
260
W
4
240
U W
220
LVV
3000
4000
3500
4500
m/z Figure 2. TOF mass spectrum of negative ions from pd(T),, laserdesorbed from a sinapinic acid (SA) matrix by 337nm light at 38 mJ/cm2resulting from summing over 100 laser pulses, showing (a) the raw data and (b) the result of 5 point smoothing of the spectrum. The molar ratio of sinapinic acid to oligomer was 2000:l.
MALD MS O F OLIGODEOXYTHYMIDYLIC ACIDS
211
2500
2400
> t m
5 t-
2300
za
2200
z
W
J
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[r
2100
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3000
(b)
4000
5000
6000
7000
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2300
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2000 3000
E 3500
4
4000
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2100
Figure3. TOF mass spectrum of negative ions from pd(T),, laserdesorbed from a DHBA matrix by 337 nm light at 62 mJ/cm2 resulting from summing over 200 laser pulses, showing (a) the raw data and (b) the result of 5 point smoothing of the spectrum. The molar ratio of DHBA to oligomer was 6000:l.
as the deprotonated unfragmented negative ion of the analyte. The substitution of a proton by a sodium ion can be observed, probably occurring in a phosphate group in the backbone of the 01igomer.~ The unidentified adduct feature around 3730 Da, the matrix adduct peak, and the broad unresolved background bump between 3100 and 4700 Da were found to be much smaller in a few 'good' spots on a similarly prepared sample, as depicted in Fig. 4. Here the analyte with no sodium attachment and the single-sodiumexchanged molecule dominate the spectrum, most 800
[M-2HtNa]I
[M-H1-ll
qnn L""
3000
3500
2200
4000
4500
m/z
Figure4. TOF mass spectrum of negative ions from pd(T),* laserdesorbed out of a DHBA matrix by 337nm light at 62mJ/cm2, resulting from summing over 200 laser pulses (raw data). A similarly prepared sample and the same desorption conditions as in Fig. 3 were used.
2000
'
3000
4000
5000
6000
I 7000
m/z
Figure5 TOF mass spectrum of negative ions from a mixture of pd(T),, through pd(T)IBlaser-desorbed from (a) a DHBA matrix resulting from summing over 50 laser pulses, and (b) a sinapinic acid matrix resulting from over lo00 laser pulses.
likely as deprotonated species. The accuracy of the absolute mass scale of k 3 Da does not allow us to distinguish between deprotonation and electron adduction in this work, but deprotonation is already known and as the dominant mechanism for negative charging of oligonucleotides. However, the mass resolution and relative accuracy of the mass scale are high enough to show a mass difference of 22 between the main peaks instead of 23, hence proving the exchange of a proton by a sodium ion. Interestingly, only one exchange per molecule is detectable. Presuming equal probabilities for all 12 phosphate groups to undergo an exchange, a third main peak about half as intense as the other two showing two sodium atoms on the same molecule should be visible. Its absence suggests the existence of a preferred binding site of the sodium, which might be found in the phosphate at the 5' end of the chain. It is not clear why such extensive protonation occurs after starting with the sodium salt of the DNA oligomer, even if there is a preferred binding site for the sodium. This lack of sodium attachment seems fortuitous for the laserdesorption mass spectrometry, especially when compared with the case for electrospray ionization mass spectrometry where significant efforts to remove the sodium ions is required for superior mass spectra." In order to test the analysis of oligonucleotide mixtures , a mixture of 7 different oligothyminosines (pd(T),Z-pd(T)lR)was desorbed from a DHBA matrix at a total molar ratio of 10 000 :1 and a 337 nm laser
MALD MS O F OLIGODEOXYTHYMIDYLIC ACIDS
212
130 >-
f= v)
gz
120
UJ
2 I-
Is 3
110
100
2400 >-
‘“i‘“
rv)
5
5w iz 4 w
2300 2200
a 2100
2000 3000
4000
.
5000
6000
7000
m/z Figure6. Smoothed versions of Fig. 5(a) and (b), with 15 point smoothing for part (a) and 5 point smoothing for part (b).
energy density of 52mJ/cm2. The results (Fig. 5(a)) show 7 equally spaced peaks at the expected masses, rising by a factor of 2 to 4 above a broad background hump probably consisting of unresolved fragment and adduct peaks with substantial metastability. Only the first peak (pd(T),J shows no fragmentation, which appears to increase with increasing length of the molecule. For comparison, the same mixture of pd(T),, to pd(T),* was desorbed from a sinapinic acid matrix at a molar ratio of 1OOOO:l shown in Fig. 5(b). All seven peaks are distinguishable again, but the unresolved background is more substantial in this case. No fine structure is revealed although the data are averaged over 1000 laser pulses, instead of 50 in Fig. 5(a). Figure 6(a) and (b) show the data of Fig. 5 with some smoothing. In general, the ferulic acid and sinapinic acid matrices show not only greater propensity for adduct formation with the oligomers, but also considerable peak broadening. This broadening may be from one or more of several sources. It may be due to ion formation over as much as several hundred micrometers above the sample surface as a result of ion/molecule reactions from a high density expansion, or possibly a large matrix-analyte cluster emitting molecules over a similarly significant distance above the surface. Both of these situations would lead to very significant kinetic energy spreads. Metastable decomposition in the extraction region of the mass spectrometer similarly will result in significant peak broadening.” In contrast, the kinetic energy spread expected from the vapor expansion extrapolated from a previous measurement of desorbed neutral gramicidin S from ferulic acid matrixL3would suggest only a few eV kinetic energy
spread in this mass range which should be compensated by the reflector. Local charging on relatively thick insulating samples could conceivably also produce broadening of the mass spectrum due to variations in the electric potential at the point(s) of ion creation; however we have no reason to believe that this is responsible for the observed differences between the different matrices or on ,different locations on a given matrix because of the generally consistent thicknesses of the samples. Another obvious difficulty encountered especially with the DHBA matrix for these DNA oligomers is the nonuniformity of sample. An improved mass spectrum (Fig. 4 vs Fig. 3) was obtained for this system simply by finding an optimum location on the sample under the desorbing laser beam even though considerable care had gone into obtaining uniform crystallization. In fact, cleavage of a relatively large single crystal of the DHBA-oligomer system with a razor blade and subsequent analysis just resulted in mass spectra like the one shown in Fig. 3. At this time, we do not know what exactly is responsible for producing an optimum vs nonoptimum location on the sample in terms of different crystallization. Furthermore, finding these optimum locations currently is simply a matter of scanning the sample position under the laser beam with an x-y manipulator and observing the mass spectrum in realtime on an oscilloscope until an apparent ‘good spot’ is found; this obviously is an undesirable approach in the long run. CONCLUSION Three aromatic organic acids have been examined as matrices for matrix-assisted UV laser desorptionl ionization of synthetic thyminosine oligomers pd(T)12 to pd(T),, used as test species for single-stranded DNA. The DHBA matrix proved to produce the best mass spectra, allowing resolution of salt adducts and very clear identification of the components of a mixture of oligomers from a 12-mer to an l&mer. Relatively little sodium adduction is observed. The ferulic acid and sinapinic acid matrices suffered from significant adduct formation and peak broadening. These mass spectra show that the matrix-assisted laser desorption method holds significant promise as a future technology to directly replace gel electrophoresis in DNA sequencing. However, improvement will be required in extending the mass range by at least an order of magnitude which currently appears to be limited in these instances due to fragmentation. Major improvements in the matrix-assisted laser desorption mass spectrometry method might lie in finding alternative matrix substances and/or conditions in order to’ produce even more favorable ionization conditions. Acknowledgements The authors thank Dr C. Green for helpful discussions. Financial support from NIH (Grant No. HG00174-02) and Deutsche Forschungsgemeinschaft (fellowship for THF) is gratefully acknowledged.
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