223 GATA 8(8): 223-229, 1991
REVIEW
Applications of Mass Spectrometry to DNA Sequencing K. B R U C E J A C O B S O N , H E I N R I C H F. A R L I N G H A U S , M I C H E L L E V. B U C H A N A N , CHUNG-HSUAN CHEN, G A R Y L. G L I S H , R O B E R T L. H E T T I C H , and S C O T T A. M c L U C K E Y
The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.
ploy the Sanger procedure [1] to convert a given oligonucleotide to a series of molecules that differ in length by one nucleotide. Other methods could equally well be used. In this method, the DNA segment whose sequence is to be determined is used as a template for the enzyme DNA polymerase. When an oligonucleotide that contains a sequence that binds to a complementary sequence on the template is used as a primer, this enzyme will use the deoxynucleoside triphosphates of A, G, C, and T to extend the primer and replicate the sequence of the template DNA. Furthermore, this enzyme will be allowed to use one of the unnatrual dideoxynucleoside triphosphates of the same four bases to terminate the newly formed DNA. The enzymatically synthesized DNA molecules each contain the original primer, a replicated sequence of part of the DNA of interest, and the dideoxy terminator. In this way, a set of DNA molecules is produced that contain the primer and differ in length from each other by one nucleotide residue. Such a set of DNAs, some with a special label and some with no label, is used in all the sequencing methods to be described. The material presented below represents initial experiments, progress reports, or proposed methods for using mass spectrometry for DNA sequence analysis. In some cases, feasibility studies have shown promise with oligonucleotides of 99% to eliminate contaminating 32S. Isotopes that are < 1% abundant are quite expensive to obtain at 99% enrichment; even when 365 is purified 100 X it contains as much or more 348 as it does 365. From reports of this project at meetings, feasibility studies have demonstrated that isobaric contaminants are not a serious problem at the sensitivities tested and also that 36S-labeled reagents are available at an affordable price (T. Brennan, personal communication, DNA Sequencing Conference, 1990).
Tin and Iron Isotopes Jacobson et al. [3] are developing methods employing the stable isotopes of tin, iron, and rare earths for DNA sequencing. Multiple labels allow more DNAs to be run in the electrophoresis gel simultaneously and thus economize on the time and cost of this step in sequence analysis. The four isotopes of sulfur, mentioned above, and the four of iron allow a modest multiplexing. The ten isotopes of tin allow more multiplexing and the many isotopes of the lanthanides would make the multiplex method even more powerful. In addition to the economic advantages, the combination of the A, G, C, and T termination types of the Sanger procedure into one lane will increase the accuracy of reading the pattern. Methods were devised from the literature to convert the oxides of tin and iron, the forms in which
isotopes are usually supplied, to triethylstannylpropanoic acid and ferrocene carboxylic acid, respectively [3, 4]. The N-hydroxysuccinimide ester of each of these organometallic reagents was allowed to react with the amino group of a 5'-hexylamine that was attached in the DNA synthesizer to the 17-mer oligonucleotide that contained the M13 DNA primer sequence. Since the primer is labeled with triethylstannylpropanamide and it becomes an integral part of the DNAs produced in the Sanger procedure, there is an atom of the metal isotope attached to each DNA. After gel electrophoresis, the DNA bands on the gel slabs can be located on the dried gel by sputterinitiated resonance ionization spectroscopy (SIRIS) [3-6]. At each given location on the gel, some of the material is sputtered off using an ion beam. This process releases some of the metal from the label as neutral atoms. The selective event for SIRIS is then the elevation of a ground-state electron of the label element to an excitation-energy state by means of carefully tuned laser beams. Subsequent ionization of the excited atom by the same or a different laser beam results in the formation of a positively charged metal ion that can be quantitatively measured by a magnetic sector mass spectrometer, or a time-of-flight mass spectrometer when several isotopes are to be measured simultaneously. Laser beams of carefully chosen wavelengths [5, 6], each unique to the chosen element, are used to accomplish the resonance ionization. To detect tin- or iron-labeled DNA on dried gels, a pulsed argon ion beam, whose diameter can be varied from 5 txm to 2 mm, is focused on the gel to atomize material on the surface. Sputtered ions produced by the ion beam are removed by an extraction electrode prior to the application of the resonance laser beams. The remaining cloud of neutral atoms and molecular fragments is illuminated by pulsed laser beams that are tuned to the element of interest so as to cause resonant ionization. These ions are then accelerated into the mass spectrometer for quantitation. We have demonstrated that, when the resonance lasers were turned off, serious interference by isobaric molecular fragments occurs in the mass regions for tin (112-124 mass units [mu]) and for iron (5458 mu). This indicates that SIMS (secondary ion mass spectrometry), a simpler form of mass spectrometry than SIRIS, may not be useful for this type of analysis. On the other hand, a high mass resolution spectrometer, used in the SIMS mode, could be useful. A high rate of DNA sequencing is potentially available since the SIRIS can be operated at 600010,000 analyses per second and, thus, the analysis
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225 Mass Spectrometry for DNA Sequencing
of 500 DNA bands, corresponding to all the fragments from one DNA segment that are labeled using four different isotopes of tin, on a 10-cm gel can be calculated to occur in - 3 s [3]. By using eight isotopes of tin, for the sequencing of two different DNA segments simultaneously, this rate can be doubled. Two problems have been encountered with the SIRIS method: (a) The ion beam, used to atomize the sample, causes accumulation of a charge that disrupts the analysis. This problem was solved by releasing low-energy electrons onto the sample between the analytic pulses. The charge from the ion beam was dissipated and no longer interfered [3]. (b) The ion beam atomizes material only in the surface monolayer. To penetrate into the surface even a few monolayers requires hundreds of pulses of the ion beam. Since the dried gel is - 1 0 - 4 0 Ixm thick, only a small fraction of the labeled DNA is potentially reached by the ion beam. However, the distribution of the DNA in the gel was found to be nonuniform in that the Sn-labeled DNA in the surface monolayer was much more concentrated than it was a few nanometers down into the gel [6]. This distribution has allowed detection of Sn-labeled DNA on polyacrylamide gels under a variety of conditions [3-6] and especially from a Sanger sequencing gel [4]. Further development of this method will focus on ways to increase the amount of DNA that is on a surface and available for analysis. An alternative to SIRIS is laser atomization RIS (LARIS) where a laser beam replaces the ion beam used to atomize the gel surface. The advantage of LARIS is that the beam is more energetic and removes much more sample material per pulse. On the other hand, the LARIS technique has received much less attention than SIRIS and additional developmental studies are necessary to prove its efficacy. Comparative evaluation of the SIRIS and LARIS techniques is under way [6].
Direct Mass Spectral Analysis of Large DNA Oligomers While much progress has been made with the ionization and subsequent mass spectral analysis of large biopolymers such as proteins and peptides, the success with intact DNA oligomers has been much more limited. In particular, oligonucleotides are very polar and are difficult to volatilize [7]. For these biomolecules, fragmentation can readily occur at the labile glycosidic bond, which detaches the nucleic acid bases from the phosphodiester backbone and destroys all sequence information. Thus, ionization techniques
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that have been used successfully for the investigation of peptides and other biopolymers have had only limited utility for oligonucleotides. The generation of molecular information from desorption techniques such as secondary ion mass spectrometry [8], fast atom bombardment [9], and plasma desorption [10] have been limited, in general, to oligomers having eight or fewer bases. In addition, the irreproducibility of molecular ion formation and poor sensitivities have limited the application of these techniques. Recent advances in the generation and detection of even larger ions (Mr = >300,000) from proteins by using matrix-assisted laser desorption and electrospray ionization have prompted a number of research groups to devise novel methods to sequence DNA oligomers directly by using these two ionization techniques. A few general approaches that use these ionization techniques for DNA sequencing are presented here. In fact, this very active research area is just in its initial stages and few open-literature publications have appeared. Thus, the citations given are not meant to be exhaustive, but rather representative of a cross section of ongoing programs that use these two ionization techniques for DNA characterization.
Matrix-Assisted Laser Desorption Karas and coworkers [11-13] first reported matrixassisted laser desorption time-of-flight mass spectrometry for the generation of ions from proteins. The biomolecules were mixed in aqueous solution with an excess of matrix such as nicotinic acid. When the laser beam (266 nm for nicotinic acid) is focused onto the dried sample, the laser energy is primarily absorbed by the matrix material, which vaporizes and carries the interspersed analyte (both neutrals and ions) into the gas phase. This technique is relatively simple and produces primarily the molecule with a proton attached ([M + H ] ÷) for proteins up to Mr>400,000, although (M +2H) 2÷ is also seen in some cases. Laser power density is critical in this experiment and must be kept in the range of 106-107 W/cm 2. Matrices other than nicotinic acid have been successfully used for these experiments, but the laser wavelength is changed to correspond to the optimum absorption of the matrix. To apply matrix-assisted laser desorption to DNA sequencing, an array of oligonucleotides must be produced by using the Sanger procedure, followed by direct detection of the oligonucleotide mixture by mass spectrometry. DNA sequencing by this approach has the potential of being much faster and
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simpler than approaches that use gel electrophoresis. Its success, however, hinges on the ability to avoid fragmentation of the individual oligomers and to produce, in every case, a singly-charged species for each oligomer with an intensity representative of its concentration in the sample. Detection limits in the femtomole-picomole region have been reported for this technique [14]. In fact, the area of the dried sample actually profiled by the laser desorption pulse is usually much smaller than the sample coverage on the probe, indicating that the amount of samples consumed per laser short is in the low-attomole region. A number of groups have been actively pursuing the analyses of large oligonucleotides using matrixassisted techniques, including Hillenkamp et al. [ 14], Beavis and Chait [15], Chait et al. [16], Cotter and coworkers [17, 18], and Hettich and Buchanan [19]. To date, the largest oligonucleotide observed intact by matrix-assisted laser desorption is a 77-mer (Mr = - 25,000) by Hillenkamp et al. [14]. Many of the reported studies on oligonucleotides have used nicotinic acid as the matrix, which absorbs at 266 nm. However, this wavelength is near the absorption optimum for nucleosides and it causes fragmentation. Beavis and Chait [20] have demonstrated that the use of sinapinic acid as a matrix with radiation at 355 nm may be a more suitable matrix for oligonucleotides. Levis and Romano [21] reported that rhodamine 6G, which absorbs green light, was useful for mass spectrometry of single-stranded DNA. A variety of matrix-wavelength combinations have been reported for the matrix assisted laser desorption conditions and, when optimized, may provide much better results for oligonucleotides. Several basic criteria are used initially to choose a suitable matrix compound: (a) the matrix must have strong spectral absorption at the chosen laser wavelength, (b) the matrix must be compatible with the analyte, and (c) the matrix must not produce ions that interfere with ions from the analyte. Detailed examinations with a variety of matrix compounds have indicated that other variables, many of which are currently unknown, also contribute to the matrix enhancement process. Although most of the matrix-assisted laser desorption spectra of oligonucleotides have been generated on time-of-flight instruments, Hettich and Buchanan [19] have used a Fourier transform mass spectrometer (FTMS) for detection of the desorbed ions. This instrument has very high resolving power and could be very useful for resolving the many oligonucleotides that result from the Sanger method. In addition, the accurate mass measurement and
multistage mass spectrometry capabilities of this instrument have been used to examine the collisioninduced dissociation (MS/MS) products of oligonucleotide ( M - H ) - ions. Preliminary experiments indicate that more fragmentation is observed in the FTMS spectra relative to the time-of-flight spectra. Although this information is quite useful for examining the structures of small oligonucleotides, the presence of fragment ions will complicate the examination of oligonucleotide mixtures. Experiments are currently under way to investigate the nature of the FTMS technique, reduce the amount of fragmentation, and increase the useable mass range [22]. Williams and colleagues [23, 24] (personal communication, 1991), who have examined visible laser desorption of a frozen aqueous matrix of oligonucleotides and DNA, observed enhanced production of molecular positive ions when the laser was tuned to a frequency corresponding to a resonant electronic transition in sodium atoms. Molecular ions of singlestranded DNA were observed at masses up to m/z 17,900. Desorption processes often result in the production of more neutral molecules than ions for most compounds. However, because DNA oligomers are very polar, the percentage of ions produced by the desorption process alone can be significant. Other types of schemes have been used to ionize neutral oligonucleotide molecules that desorb as a result of laser irradiation of the sample. Linder et al. [26] as well as Grotemeyer [26] have used multiphoton ionization to generate positive ions of small oligonucleotides desorbed as intact molecules with an infrared laser (CO2). After laser desorption, the neutral molecules were entrained in an expanding supersonic stream of argon. Fragmentation was reduced due to the cooling in the nozzle expansion; however, the ability to efficiently multiphoton ionize these oligonucleotides decreases as they become larger. Huth-Fehre and Becker [27] have used a vacuum ultraviolet ionization following laser desorption from a ferulic acid matrix to generate primarily molecular ions. Recently, Chen and colleagues [28] have begun experiments to attach ferrocene to oligomers to reduce the ionization threshold and then use laser energies just above this threshold to produce ions from laser-desorbed neutrals without producing fragment ions. In summary, matrix-assisted laser desorption is a very promising approach for sequencing DNA oligomers. In combination with the Sanger process, it has the potential of being able to determine the Sanger segments rapidly from mixtures with no prior separation. For this approach to be successful, a bet-
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227 Mass Spectrometry for DNA Sequencing
ter understanding of the fundamentals of the desorption mechanism for both ions and neutrals is required. This must include the investigation of numerous matrices and wavelength combinations to control both desorption and fragmentation.
Electrospray Ionization Another technique that indicates significant potential for the direct mass spectral examination of large biomolecules is electrospray ionization, which is accomplished by applying a high voltage (3-5 kV) to a flow of liquid (0.5-50 ~L/min) exiting a small needle or capillary. This is executed at atmospheric pressure and generates small, highly charged droplets that desolvate as they traverse from the needle to an aperture or capillary that serves as an interface between atmospheric pressure and the vacuum of the mass spectrometer. In contrast to matrix-assisted laser desorption, which produces predominantly singly-charged ions (up to m/z 400,000 or higher), electrospray produces ions with multiple charges, primarily in the m/z 500-2000 region, for these large biomolecules. This "folding back" of the spectrum of the large biomolecules has the advantage of allowing standard instruments (for example, quadrupole filters, sectors, quadrupole ion trap, and FTMS) to be used for the analysis. In addition, because multiple charge states (z) are generated for each compound (m), a range of mass-to-charge (m/z) ratios (that is, the multiple peaks in the electrospray mass spectrum) may be measured. Because m is constant and z varies, enhanced precision in determining the molecular mass is obtained by averaging all of the measured mass values determined from the various rn/z ratios (provided that the charge states can be distinguished). A serious drawback of the formation of multiple m/z ratios, however, is that the analysis of impure samples can result in very complicated spectra from many ions with overlapping m/z ratios. This suggests that direct analysis of an oligonucleotide mixture generated by the Sanger method will not be feasible with electrospray. Because of the low flow rates, however, capillary electrophoresis combined with electrospray ionization is reasonable and may be applicable to DNA sequencing, but this approach will ultimately be slower than techniques that do not rely on separation techniques, such as matrix-assisted laser desorption, prior to mass spectral detection. Like matrix-assisted laser desorption, electrospray has been widely applied to the analysis of peptides and proteins [29] and indicates detection limits in the femtomole-picomole range, but less work has been
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reported for oligonucleotides. Covey et al. [30, 31 ] first demonstrated electrospray generation of ions from a 14-base oligomer. The resulting spectrum yielded ions from ( M - 6 H ) ~ to ( M - 1 1 H ) -~. The oligonucleotides readily yield negative ions because the phosphate sites are deprotonated in solution. Molecular weights up to -25,000 have been reported for tRNA by Smith et al. [32]. These studies found that traces of sodium that occur adventitiously in the sample result in replacement of some of the protons by sodium ions, resulting in unresolved isotopic clusters for large values of z. This arises because, in addition to ions at ( M - n i l ) ~-, there are also ions at (M [ n + l ] H + Na)n-, ( M - [n+2]H + 2Na) ~, and so forth. Because sodium ion contamination occurs in both synthetic and natural oligonucleotides, this represents a serious limitation to this technique. Two very recent studies by Stults and Marsters [33,34] and Van Berkel et al. (unpublished results, 1991) have demonstrated that solution chemistry can be used to minimize this sodium ion problem. In the Stults and Marsters study [33], sodium ions were removed by precipitation and the oligonucleotides were converted to the more volatile ammonium salt. As a result, a 77-mer was found to have only one sodium in thedominant peak, while a 48-mer gave ions with no sodium present when this approach was used. Van Berkel et al. (unpublished results, 1991) have removed Na ÷ ions on line with the use of solutions containing selective complexing agents, such as crown ethers. Tandem mass spectrometry (MS/MS) of small multiply-charged oligonucleotides (8-mers) has been demonstrated by McLuckey et al. [35] using a quadrupole ion trap. These results indicate that the multiple charges make these ions very fragile and thus amenable to MS n and have demonstrated that some sequence information on small oligonucleotides can be obtained by M S 3. High selectivity for fragmentation loss of the adenine anion was observed with subsequent dissociation of the nucleotide chain at that site, regardless of the adenine sequence location. It remains to be determined how much sequence information can be obtained for oligonucleotides of unknown sequence and chain length by using MS/MS techniques, however. In summary, electrospray has the potential of generating ions from large oligonucleotides, but, for use as a DNA sequencing technique, it is most likely that separations techniques will be required prior to electrospray mass spectral detection because of the extraordinarily complex spectra that would be produced from a Sanger mixture of oligonucleotides. Clearly,
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however, this technique shows considerable promise and merits additional investigation in order to expand the upper mass of oligonucleotides that may be analyzed using this approach.
Summary A number of approaches for D N A sequencing by mass spectrometry have been proposed and more are likely to be pursued as this technology advances. Approaches that employ stable isotopes as labels on the oligomers avoid the requirement of generating high-mass ions and only detect the particular isotope label. By using several stable isotopes, four, eight, 12, or more labels can be read simultaneously in the same lane of the electrophoresis gel that separate the oligomers by molecular weight. In this manner, a definite advantage over radiolabel procedures with respect to accuracy and overall sequencing time should be obtained with this multiplexing approach. The development of new techniques for the generation of molecular ions from high-mass biomolecules has led to interest in the examination of intact DNA oligomers. Recent developments in matrix-assisted laser desorption have demonstrated that molecular (M + H) ÷ or ( M - H)- ions can be obtained for oligomers with Mr>23,000, with little fragmentation. This suggests that direct sequencing of Sanger mixtures may be possible using this technique, which would offer a considerable time advantage over sequencing methods that require separation methods. Electrospray ionization also has been used to generate spectra from oligomers with molecular weights comparable to those obtained by matrix-assisted laser desorption. This technique, due to the nature of multiple peaks generated for each compound, would most likely be used for sequencing Sanger fragments only after separation by capillary gel electrophoresis. All of the approaches described in this review are in initial stages of investigation. It is quite possible that breakthroughs will be made in ionization methods, chemical conditions, and instrumentation to advance these and perhaps other mass spectrometry approaches to the point where they can be used for routine sequencing of DNA.
This research was sponsored by the ORNL Director's Research and Development Fund, the Office of Health and Environmental Research, and the Office of Basic Energy Sciences of the US Department of Energy, under contract DEAC05-84OR-21400 to Martin Marietta Energy Systems, Inc., and under contract DE-FG05-90ER-81048 to Atom Sciences, Inc.
References 1. Sanger F, Nickelen S, Coulson AR: Proc Natl Acad Sci USA 94:5463-5467,1977 2. Brennan T, Chakel J, Bente P, Field M: In Burlingame AL, McCloskey JA (eds): Biol Mass Spectroscopy. New York, Elsevier, 1990, pp 219-227 3. Jacobson KB, Arlinghaus HF, Schmitt HW, Sachleben RA, Brown GM, Thonnard N, Sloop FV, Foote RS, Larimer FW, Woychik RP, England MW, Burchett KL, Jacobson DA: Genomics 9:51-59, 1991 4. Sachleben RA, Brown GM, Sloop FV, Arlinghaus HF, England MW, Foote RS, Larimer FW, Woychik RP, Thonnard N, Jacobson KB: Genet Anal Tech Appl 8:167-170, 1991 5. Arlinghaus HF, Thonnard N, Spaar MT, Sachleben RA, Larimer FW, Foote RS, Woychik RP, Brown GM, Sloop FV, Jacobson KB: Anal Chem 63:402-407, 1991 6. Arlinghaus HF, Thonnard N, Spaar MT, Sach MT, Sachleben RA, Larimer FW, Foote RS, Woychik RP, Brown GM, Sloop AV, Peterson JR, Jacobson KB: J Vac Sci Technol A9:1312-1319, 1991 7. McCloskey JA (ed): Mass Spectrometry. Methods in Enzymology, vol 193. New York, Academic Press, 1990. 8. BenninghovenA, Riidenauer FG, Werner HW: In Secondary Ion Mass Spectroscopy. New York, John Wiley and Sons, 1987 9. Cerny RL, Gross ML, Grotjahn L: Anal Biochem 156:424435, 1986 10. McNeal CJ, Narang SA, MacFarlane RD, Hsiung HM, Bronsseau R: Proc Natl Acad Sci USA 77:735-739, 1980 ! 1. Karas M, Hillenkamp F: Anal Chem 60:2299-2301, 1988 12. Hillenkamp F, Karas M, Beavis RC, Chait BP: Anal Chem 63:1193A-1203A, 1991 13. Karas M, Ingendoh A, Bahr U, Hillenkamp, F: Biomed Environ Mass Spectrom 18:841-843, 1989 14. HillenkampF, Karas M, Ingendoh A, Stahl B: In Burlingame AL, McCloskey JA (eds): Biol Mass Spectroscopy. New York, Elsevier, 1990, pp 49-57 15. Beavis RC, Chait, BT: Rapid CommunMass Spectrom3:233237, 1989 16. Chait BT, Chowelhury SK, Katta V, Beavis RC: Proceedings of 38th ASMS Conference on Mass Spectrometry, 1990, pp 28-29 17. Spengler B, Pan Y, Cotter RJ: Anal Chem 62:793-796, 1990 18. Spengler B, Pan Y, Cotter RJ: Anal Chem 62:793-796, 1990 19. Hettich R, Buchanan M: J Am Soc Mass Spectrom 21:402412, 1991 20. Beavis RC, Chait BP: Proceedings of the 38th ASMS Conference on Mass Spectrometry and Allied Topics, Tuscon, 3-8 June 1990, pp 152-153 21. Levis RJ, Romano LJ: J Am Chem Soc 113:7802-7803, 1991 22. Hettich RL, Buchanan MV: Int J Mass Spectrom Ion Proc 111:365-380, 1991 23. Nelson RW, Rainbow MJ, Lohr DE, Williams P: Science 246:1585-1587, 1989 24. Nelson RW, Thomas RM, Williams P: Rapid Commun Mass Spectrom 4:348-351, 1990
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25. Lindner L, Grotemeyer J, Schlag EW: Int J Mass Spectrom Ion Proc 100:267-285, 1990 26. Grotemeyer J: Proceedings of the Workshop on Laser Ablation: Mechanisms and Applications: Lecture Notes in Physics. New York, Springer-Verlag 1992 (in press) 27. Huth-Fehre T, Becker CH: Rapid Commun Mass Spectrom 5:378-382,1991 28. Chen CH, Payne MG, Jacobson KB: Int J Genome Res 1992 (in press) 29. Fenn JB, Mann M, Meng CK, Wong SK: Mass Spectrom Rev 9:37-70, 1990
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30. Covey TR, Banner RF, Shushan BI, Henion J: Rapid Commun Mass Spectrom 2:249-256, 1988 31. Covey TR, Bonner RF, Shushan BI, Henion J: Rapid Commun Mass Spectrom 2:249, 1988 32. Smith RD, Loo JA, Edmonds CG, Barinaga CJ, Udseth HR: Anal Chem 62:882-899, 1990 33. StultsJT, Marsters JC: RapidCommunMass Spectrom5:54635467, 1991 34. Stults JT, Marsters JC: Rapid Commun Mass Spectrom 5:359363, 1991 35. McLuckey SA, Van Berkel GJ, Glish GL: J Am Soc Mass Spectrom 3:60-70, 1992
Published 1992 by Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010
REVIEW
Applications of Mass Spectrometry to DNA Sequencing K. B R U C E J A C O B S O N , H E I N R I C H F. A R L I N G H A U S , M I C H E L L E V. B U C H A N A N , CHUNG-HSUAN CHEN, G A R Y L. G L I S H , R O B E R T L. H E T T I C H , and S C O T T A. M c L U C K E Y
The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.
ploy the Sanger procedure [1] to convert a given oligonucleotide to a series of molecules that differ in length by one nucleotide. Other methods could equally well be used. In this method, the DNA segment whose sequence is to be determined is used as a template for the enzyme DNA polymerase. When an oligonucleotide that contains a sequence that binds to a complementary sequence on the template is used as a primer, this enzyme will use the deoxynucleoside triphosphates of A, G, C, and T to extend the primer and replicate the sequence of the template DNA. Furthermore, this enzyme will be allowed to use one of the unnatrual dideoxynucleoside triphosphates of the same four bases to terminate the newly formed DNA. The enzymatically synthesized DNA molecules each contain the original primer, a replicated sequence of part of the DNA of interest, and the dideoxy terminator. In this way, a set of DNA molecules is produced that contain the primer and differ in length from each other by one nucleotide residue. Such a set of DNAs, some with a special label and some with no label, is used in all the sequencing methods to be described. The material presented below represents initial experiments, progress reports, or proposed methods for using mass spectrometry for DNA sequence analysis. In some cases, feasibility studies have shown promise with oligonucleotides of 99% to eliminate contaminating 32S. Isotopes that are < 1% abundant are quite expensive to obtain at 99% enrichment; even when 365 is purified 100 X it contains as much or more 348 as it does 365. From reports of this project at meetings, feasibility studies have demonstrated that isobaric contaminants are not a serious problem at the sensitivities tested and also that 36S-labeled reagents are available at an affordable price (T. Brennan, personal communication, DNA Sequencing Conference, 1990).
Tin and Iron Isotopes Jacobson et al. [3] are developing methods employing the stable isotopes of tin, iron, and rare earths for DNA sequencing. Multiple labels allow more DNAs to be run in the electrophoresis gel simultaneously and thus economize on the time and cost of this step in sequence analysis. The four isotopes of sulfur, mentioned above, and the four of iron allow a modest multiplexing. The ten isotopes of tin allow more multiplexing and the many isotopes of the lanthanides would make the multiplex method even more powerful. In addition to the economic advantages, the combination of the A, G, C, and T termination types of the Sanger procedure into one lane will increase the accuracy of reading the pattern. Methods were devised from the literature to convert the oxides of tin and iron, the forms in which
isotopes are usually supplied, to triethylstannylpropanoic acid and ferrocene carboxylic acid, respectively [3, 4]. The N-hydroxysuccinimide ester of each of these organometallic reagents was allowed to react with the amino group of a 5'-hexylamine that was attached in the DNA synthesizer to the 17-mer oligonucleotide that contained the M13 DNA primer sequence. Since the primer is labeled with triethylstannylpropanamide and it becomes an integral part of the DNAs produced in the Sanger procedure, there is an atom of the metal isotope attached to each DNA. After gel electrophoresis, the DNA bands on the gel slabs can be located on the dried gel by sputterinitiated resonance ionization spectroscopy (SIRIS) [3-6]. At each given location on the gel, some of the material is sputtered off using an ion beam. This process releases some of the metal from the label as neutral atoms. The selective event for SIRIS is then the elevation of a ground-state electron of the label element to an excitation-energy state by means of carefully tuned laser beams. Subsequent ionization of the excited atom by the same or a different laser beam results in the formation of a positively charged metal ion that can be quantitatively measured by a magnetic sector mass spectrometer, or a time-of-flight mass spectrometer when several isotopes are to be measured simultaneously. Laser beams of carefully chosen wavelengths [5, 6], each unique to the chosen element, are used to accomplish the resonance ionization. To detect tin- or iron-labeled DNA on dried gels, a pulsed argon ion beam, whose diameter can be varied from 5 txm to 2 mm, is focused on the gel to atomize material on the surface. Sputtered ions produced by the ion beam are removed by an extraction electrode prior to the application of the resonance laser beams. The remaining cloud of neutral atoms and molecular fragments is illuminated by pulsed laser beams that are tuned to the element of interest so as to cause resonant ionization. These ions are then accelerated into the mass spectrometer for quantitation. We have demonstrated that, when the resonance lasers were turned off, serious interference by isobaric molecular fragments occurs in the mass regions for tin (112-124 mass units [mu]) and for iron (5458 mu). This indicates that SIMS (secondary ion mass spectrometry), a simpler form of mass spectrometry than SIRIS, may not be useful for this type of analysis. On the other hand, a high mass resolution spectrometer, used in the SIMS mode, could be useful. A high rate of DNA sequencing is potentially available since the SIRIS can be operated at 600010,000 analyses per second and, thus, the analysis
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225 Mass Spectrometry for DNA Sequencing
of 500 DNA bands, corresponding to all the fragments from one DNA segment that are labeled using four different isotopes of tin, on a 10-cm gel can be calculated to occur in - 3 s [3]. By using eight isotopes of tin, for the sequencing of two different DNA segments simultaneously, this rate can be doubled. Two problems have been encountered with the SIRIS method: (a) The ion beam, used to atomize the sample, causes accumulation of a charge that disrupts the analysis. This problem was solved by releasing low-energy electrons onto the sample between the analytic pulses. The charge from the ion beam was dissipated and no longer interfered [3]. (b) The ion beam atomizes material only in the surface monolayer. To penetrate into the surface even a few monolayers requires hundreds of pulses of the ion beam. Since the dried gel is - 1 0 - 4 0 Ixm thick, only a small fraction of the labeled DNA is potentially reached by the ion beam. However, the distribution of the DNA in the gel was found to be nonuniform in that the Sn-labeled DNA in the surface monolayer was much more concentrated than it was a few nanometers down into the gel [6]. This distribution has allowed detection of Sn-labeled DNA on polyacrylamide gels under a variety of conditions [3-6] and especially from a Sanger sequencing gel [4]. Further development of this method will focus on ways to increase the amount of DNA that is on a surface and available for analysis. An alternative to SIRIS is laser atomization RIS (LARIS) where a laser beam replaces the ion beam used to atomize the gel surface. The advantage of LARIS is that the beam is more energetic and removes much more sample material per pulse. On the other hand, the LARIS technique has received much less attention than SIRIS and additional developmental studies are necessary to prove its efficacy. Comparative evaluation of the SIRIS and LARIS techniques is under way [6].
Direct Mass Spectral Analysis of Large DNA Oligomers While much progress has been made with the ionization and subsequent mass spectral analysis of large biopolymers such as proteins and peptides, the success with intact DNA oligomers has been much more limited. In particular, oligonucleotides are very polar and are difficult to volatilize [7]. For these biomolecules, fragmentation can readily occur at the labile glycosidic bond, which detaches the nucleic acid bases from the phosphodiester backbone and destroys all sequence information. Thus, ionization techniques
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that have been used successfully for the investigation of peptides and other biopolymers have had only limited utility for oligonucleotides. The generation of molecular information from desorption techniques such as secondary ion mass spectrometry [8], fast atom bombardment [9], and plasma desorption [10] have been limited, in general, to oligomers having eight or fewer bases. In addition, the irreproducibility of molecular ion formation and poor sensitivities have limited the application of these techniques. Recent advances in the generation and detection of even larger ions (Mr = >300,000) from proteins by using matrix-assisted laser desorption and electrospray ionization have prompted a number of research groups to devise novel methods to sequence DNA oligomers directly by using these two ionization techniques. A few general approaches that use these ionization techniques for DNA sequencing are presented here. In fact, this very active research area is just in its initial stages and few open-literature publications have appeared. Thus, the citations given are not meant to be exhaustive, but rather representative of a cross section of ongoing programs that use these two ionization techniques for DNA characterization.
Matrix-Assisted Laser Desorption Karas and coworkers [11-13] first reported matrixassisted laser desorption time-of-flight mass spectrometry for the generation of ions from proteins. The biomolecules were mixed in aqueous solution with an excess of matrix such as nicotinic acid. When the laser beam (266 nm for nicotinic acid) is focused onto the dried sample, the laser energy is primarily absorbed by the matrix material, which vaporizes and carries the interspersed analyte (both neutrals and ions) into the gas phase. This technique is relatively simple and produces primarily the molecule with a proton attached ([M + H ] ÷) for proteins up to Mr>400,000, although (M +2H) 2÷ is also seen in some cases. Laser power density is critical in this experiment and must be kept in the range of 106-107 W/cm 2. Matrices other than nicotinic acid have been successfully used for these experiments, but the laser wavelength is changed to correspond to the optimum absorption of the matrix. To apply matrix-assisted laser desorption to DNA sequencing, an array of oligonucleotides must be produced by using the Sanger procedure, followed by direct detection of the oligonucleotide mixture by mass spectrometry. DNA sequencing by this approach has the potential of being much faster and
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simpler than approaches that use gel electrophoresis. Its success, however, hinges on the ability to avoid fragmentation of the individual oligomers and to produce, in every case, a singly-charged species for each oligomer with an intensity representative of its concentration in the sample. Detection limits in the femtomole-picomole region have been reported for this technique [14]. In fact, the area of the dried sample actually profiled by the laser desorption pulse is usually much smaller than the sample coverage on the probe, indicating that the amount of samples consumed per laser short is in the low-attomole region. A number of groups have been actively pursuing the analyses of large oligonucleotides using matrixassisted techniques, including Hillenkamp et al. [ 14], Beavis and Chait [15], Chait et al. [16], Cotter and coworkers [17, 18], and Hettich and Buchanan [19]. To date, the largest oligonucleotide observed intact by matrix-assisted laser desorption is a 77-mer (Mr = - 25,000) by Hillenkamp et al. [14]. Many of the reported studies on oligonucleotides have used nicotinic acid as the matrix, which absorbs at 266 nm. However, this wavelength is near the absorption optimum for nucleosides and it causes fragmentation. Beavis and Chait [20] have demonstrated that the use of sinapinic acid as a matrix with radiation at 355 nm may be a more suitable matrix for oligonucleotides. Levis and Romano [21] reported that rhodamine 6G, which absorbs green light, was useful for mass spectrometry of single-stranded DNA. A variety of matrix-wavelength combinations have been reported for the matrix assisted laser desorption conditions and, when optimized, may provide much better results for oligonucleotides. Several basic criteria are used initially to choose a suitable matrix compound: (a) the matrix must have strong spectral absorption at the chosen laser wavelength, (b) the matrix must be compatible with the analyte, and (c) the matrix must not produce ions that interfere with ions from the analyte. Detailed examinations with a variety of matrix compounds have indicated that other variables, many of which are currently unknown, also contribute to the matrix enhancement process. Although most of the matrix-assisted laser desorption spectra of oligonucleotides have been generated on time-of-flight instruments, Hettich and Buchanan [19] have used a Fourier transform mass spectrometer (FTMS) for detection of the desorbed ions. This instrument has very high resolving power and could be very useful for resolving the many oligonucleotides that result from the Sanger method. In addition, the accurate mass measurement and
multistage mass spectrometry capabilities of this instrument have been used to examine the collisioninduced dissociation (MS/MS) products of oligonucleotide ( M - H ) - ions. Preliminary experiments indicate that more fragmentation is observed in the FTMS spectra relative to the time-of-flight spectra. Although this information is quite useful for examining the structures of small oligonucleotides, the presence of fragment ions will complicate the examination of oligonucleotide mixtures. Experiments are currently under way to investigate the nature of the FTMS technique, reduce the amount of fragmentation, and increase the useable mass range [22]. Williams and colleagues [23, 24] (personal communication, 1991), who have examined visible laser desorption of a frozen aqueous matrix of oligonucleotides and DNA, observed enhanced production of molecular positive ions when the laser was tuned to a frequency corresponding to a resonant electronic transition in sodium atoms. Molecular ions of singlestranded DNA were observed at masses up to m/z 17,900. Desorption processes often result in the production of more neutral molecules than ions for most compounds. However, because DNA oligomers are very polar, the percentage of ions produced by the desorption process alone can be significant. Other types of schemes have been used to ionize neutral oligonucleotide molecules that desorb as a result of laser irradiation of the sample. Linder et al. [26] as well as Grotemeyer [26] have used multiphoton ionization to generate positive ions of small oligonucleotides desorbed as intact molecules with an infrared laser (CO2). After laser desorption, the neutral molecules were entrained in an expanding supersonic stream of argon. Fragmentation was reduced due to the cooling in the nozzle expansion; however, the ability to efficiently multiphoton ionize these oligonucleotides decreases as they become larger. Huth-Fehre and Becker [27] have used a vacuum ultraviolet ionization following laser desorption from a ferulic acid matrix to generate primarily molecular ions. Recently, Chen and colleagues [28] have begun experiments to attach ferrocene to oligomers to reduce the ionization threshold and then use laser energies just above this threshold to produce ions from laser-desorbed neutrals without producing fragment ions. In summary, matrix-assisted laser desorption is a very promising approach for sequencing DNA oligomers. In combination with the Sanger process, it has the potential of being able to determine the Sanger segments rapidly from mixtures with no prior separation. For this approach to be successful, a bet-
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227 Mass Spectrometry for DNA Sequencing
ter understanding of the fundamentals of the desorption mechanism for both ions and neutrals is required. This must include the investigation of numerous matrices and wavelength combinations to control both desorption and fragmentation.
Electrospray Ionization Another technique that indicates significant potential for the direct mass spectral examination of large biomolecules is electrospray ionization, which is accomplished by applying a high voltage (3-5 kV) to a flow of liquid (0.5-50 ~L/min) exiting a small needle or capillary. This is executed at atmospheric pressure and generates small, highly charged droplets that desolvate as they traverse from the needle to an aperture or capillary that serves as an interface between atmospheric pressure and the vacuum of the mass spectrometer. In contrast to matrix-assisted laser desorption, which produces predominantly singly-charged ions (up to m/z 400,000 or higher), electrospray produces ions with multiple charges, primarily in the m/z 500-2000 region, for these large biomolecules. This "folding back" of the spectrum of the large biomolecules has the advantage of allowing standard instruments (for example, quadrupole filters, sectors, quadrupole ion trap, and FTMS) to be used for the analysis. In addition, because multiple charge states (z) are generated for each compound (m), a range of mass-to-charge (m/z) ratios (that is, the multiple peaks in the electrospray mass spectrum) may be measured. Because m is constant and z varies, enhanced precision in determining the molecular mass is obtained by averaging all of the measured mass values determined from the various rn/z ratios (provided that the charge states can be distinguished). A serious drawback of the formation of multiple m/z ratios, however, is that the analysis of impure samples can result in very complicated spectra from many ions with overlapping m/z ratios. This suggests that direct analysis of an oligonucleotide mixture generated by the Sanger method will not be feasible with electrospray. Because of the low flow rates, however, capillary electrophoresis combined with electrospray ionization is reasonable and may be applicable to DNA sequencing, but this approach will ultimately be slower than techniques that do not rely on separation techniques, such as matrix-assisted laser desorption, prior to mass spectral detection. Like matrix-assisted laser desorption, electrospray has been widely applied to the analysis of peptides and proteins [29] and indicates detection limits in the femtomole-picomole range, but less work has been
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reported for oligonucleotides. Covey et al. [30, 31 ] first demonstrated electrospray generation of ions from a 14-base oligomer. The resulting spectrum yielded ions from ( M - 6 H ) ~ to ( M - 1 1 H ) -~. The oligonucleotides readily yield negative ions because the phosphate sites are deprotonated in solution. Molecular weights up to -25,000 have been reported for tRNA by Smith et al. [32]. These studies found that traces of sodium that occur adventitiously in the sample result in replacement of some of the protons by sodium ions, resulting in unresolved isotopic clusters for large values of z. This arises because, in addition to ions at ( M - n i l ) ~-, there are also ions at (M [ n + l ] H + Na)n-, ( M - [n+2]H + 2Na) ~, and so forth. Because sodium ion contamination occurs in both synthetic and natural oligonucleotides, this represents a serious limitation to this technique. Two very recent studies by Stults and Marsters [33,34] and Van Berkel et al. (unpublished results, 1991) have demonstrated that solution chemistry can be used to minimize this sodium ion problem. In the Stults and Marsters study [33], sodium ions were removed by precipitation and the oligonucleotides were converted to the more volatile ammonium salt. As a result, a 77-mer was found to have only one sodium in thedominant peak, while a 48-mer gave ions with no sodium present when this approach was used. Van Berkel et al. (unpublished results, 1991) have removed Na ÷ ions on line with the use of solutions containing selective complexing agents, such as crown ethers. Tandem mass spectrometry (MS/MS) of small multiply-charged oligonucleotides (8-mers) has been demonstrated by McLuckey et al. [35] using a quadrupole ion trap. These results indicate that the multiple charges make these ions very fragile and thus amenable to MS n and have demonstrated that some sequence information on small oligonucleotides can be obtained by M S 3. High selectivity for fragmentation loss of the adenine anion was observed with subsequent dissociation of the nucleotide chain at that site, regardless of the adenine sequence location. It remains to be determined how much sequence information can be obtained for oligonucleotides of unknown sequence and chain length by using MS/MS techniques, however. In summary, electrospray has the potential of generating ions from large oligonucleotides, but, for use as a DNA sequencing technique, it is most likely that separations techniques will be required prior to electrospray mass spectral detection because of the extraordinarily complex spectra that would be produced from a Sanger mixture of oligonucleotides. Clearly,
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however, this technique shows considerable promise and merits additional investigation in order to expand the upper mass of oligonucleotides that may be analyzed using this approach.
Summary A number of approaches for D N A sequencing by mass spectrometry have been proposed and more are likely to be pursued as this technology advances. Approaches that employ stable isotopes as labels on the oligomers avoid the requirement of generating high-mass ions and only detect the particular isotope label. By using several stable isotopes, four, eight, 12, or more labels can be read simultaneously in the same lane of the electrophoresis gel that separate the oligomers by molecular weight. In this manner, a definite advantage over radiolabel procedures with respect to accuracy and overall sequencing time should be obtained with this multiplexing approach. The development of new techniques for the generation of molecular ions from high-mass biomolecules has led to interest in the examination of intact DNA oligomers. Recent developments in matrix-assisted laser desorption have demonstrated that molecular (M + H) ÷ or ( M - H)- ions can be obtained for oligomers with Mr>23,000, with little fragmentation. This suggests that direct sequencing of Sanger mixtures may be possible using this technique, which would offer a considerable time advantage over sequencing methods that require separation methods. Electrospray ionization also has been used to generate spectra from oligomers with molecular weights comparable to those obtained by matrix-assisted laser desorption. This technique, due to the nature of multiple peaks generated for each compound, would most likely be used for sequencing Sanger fragments only after separation by capillary gel electrophoresis. All of the approaches described in this review are in initial stages of investigation. It is quite possible that breakthroughs will be made in ionization methods, chemical conditions, and instrumentation to advance these and perhaps other mass spectrometry approaches to the point where they can be used for routine sequencing of DNA.
This research was sponsored by the ORNL Director's Research and Development Fund, the Office of Health and Environmental Research, and the Office of Basic Energy Sciences of the US Department of Energy, under contract DEAC05-84OR-21400 to Martin Marietta Energy Systems, Inc., and under contract DE-FG05-90ER-81048 to Atom Sciences, Inc.
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