Anal. Chem. 1997, 69, 919-925
DNA Analysis Using an Electrospray Scanning Mobility Particle Sizer Ste´phane Mouradian,†,§ Jeffrey W. Skogen,‡,⊥ Frank D. Dorman,‡ Fahimeh Zarrin,‡,| Stanley L. Kaufman,‡ and Lloyd M. Smith*,†
Department of Chemistry, University of Wisconsin, Madison, 1101 University Avenue, Madison, Wisconsin 53706-1396, and TSI Incorporated, 500 Cardigan Road, St. Paul, Minnesota 55126-3996
A scanning mobility particle sizer (SMPS) allows size separation of gas phase particles according to their electrophoretic mobilities. The addition of an electrospray source (ES) recently allowed extension of SMPS analysis to the macromolecular range. We demonstrate here the application of ES-SMPS to nucleic acids analysis. Singleand double-stranded DNA molecules ranging from 6.1 kDa (single-stranded DNA 20 nucleotides in length) to 300 kDa (500 base-pair double-stranded DNA) were separated and detected by ES-SMPS at the picomole to femtomole levels. The measured electrophoretic mobility diameters were found to correlate with the analytes’ molecular weights, while the peak areas could yield quantitative information. No fragmentation of DNA was observed under the conditions employed. Different apparent densities were observed for single-stranded and double-stranded DNAs, showing a different behavior for each type of biomolecule. The total analysis time was about 3 min/spectrum. Further optimization of ES-SMPS is expected to make it a fast and sensitive technique for biopolymer characterization. Methods to analyze deoxyribonucleic acid (DNA) have received increasing attention in the past few years.1,2 A range of techniques are currently used for separations of single-stranded and double-stranded molecules. Slab gel electrophoresis using agarose1,3,4 is the most widely used methodology for the analysis of double-stranded DNA. Pulsed field gel electrophoresis in agarose allows the separation of molecules up to several million base pairs in length.5-9 Single-stranded DNA molecules are †
University of Wisconsin. TSI Inc. § Current address: Imation, 8124 Pacific Ave., White City, OR 97503. ⊥ Current address: 624 Erie St. SE, Apt #2, Minneapolis, MN 55414. | Current address: 3312 W. Burgundy Ct., Mequon, WI 53092. (1) Smith, L. M. Science 1993, 262, 530-532. (2) Hunkapiller, T.; Kaiser, R. J.; Koop, B. F.; Hood, L. E. Science 1991, 254, 59-67. (3) Atwood, T. K.; Nelmes, B. J.; Sellen, D. B. Biopolymers 1988, 27, 201. (4) Ruan, J. Z.; Litt, M. H.; Krieger, I. M. J. Colloid Interface Sci. 1988, 126, 93-106. (5) Schwartz, D. C.; Cantor, C. R. Cell 1984, 37, 67-75. (6) Turmel, C.; Brassard, E.; Foosyth, R.; Hood, K.; Slater, G. W.; Noolandi, J. Electrophoresis of Large DNA Molecules: Theory and Application; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1990; pp 101-131. (7) Turmel, C.; Brassard, E.; Slater, G. W.; Noolandi, J. Nucleic Acids Res. 1990, 18, 569-575. (8) Birren, B. W.; Lai, E.; Hood, L. E.; Simon, M. Anal. Biochem. 1989, 177, 282-286. (9) Daniels, D. L.; Olson, C. H.; Brumley, R. L.; Blattner, F. R. Nucleic Acids Res. 1990, 18, 1312. ‡
S0003-2700(96)00785-8 CCC: $14.00
© 1997 American Chemical Society
commonly separated and sequenced in polyacrylamide gels.1,10-12 Conventional slab gel electrophoresis techniques are, however, characterized by fairly lengthy run times (typically several hours).1 Capillary electrophoresis13-17 and ultrathin slab gel electrophoresis18,19 allow shorter analysis times as the separation is performed at a greater voltage. However, both electrophoresis geometries can suffer from labor-intensive gel preparation.1 To address these issues, several approaches are being investigated as alternatives to gel electrophoresis-based methodologies. Recent advances in mass spectrometry have made possible the mass analysis of large biopolymers. In matrix-assisted laser desorption/ionization (MALDI) mass spectrometry,20,21 routine analysis of DNA is, however, limited to single-stranded molecules up to about 100 base pairs in length.22-39 Fragmentation of the analyte is thought to be partly (10) Maniatis, T.; Jeffrey, A.; VandeSande, J. H. Biochemistry 1975, 14, 3787. (11) Maniatis, T.; Efstratiadis, A. Methods Enzymol. 1980, 65, 299-319. (12) Pinder, J. C.; Staynov, D. E.; Gratzer, W. B. Biochemistry 1973, 13, 53675373. (13) Luckey, J. A.; Drossman, H.; Kostichka, A. J.; Mead, D. A.; D’Cunha, J.; Norris, T. B.; Smith, L. M. Nucleic Acids Res. 1990, 18, 4417-4421. (14) Luckey, J. A.; Smith, L. M. Anal. Chem. 1993, 65, 2841-2850. (15) Luckey, J. A.; Norris, T. B.; Smith, L. M. J. Phys. Chem. 1993, 97, 30673075. (16) Karger, B. L. Nature 1989, 339, 641-642. (17) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; D’Cunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903. (18) Brumley, R. L.; Smith, L. M. Nucleic Acids Res. 1991, 19, 4121-4126. (19) Kostichka, A. J.; Marchbanks, M. L.; Brumley, R. L.; Drossman, H.; Smith, L. M. Biotechnology 1992, 10, 78-81. (20) Hillenkamp, F.; Karas, M. Anal. Chem. 1988, 60, 2301-2303. (21) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion. Processes 1987, 78, 53-68. (22) Fitzgerald, M. C.; Zhu, L.; Smith, L. M. Rapid. Commun. Mass Spectrom. 1993, 7, 895-897. (23) Parr, G. R.; Fitzgerald, M. C.; Smith, L. M. Rapid. Commun. Mass Spectrom. 1992, 6, 369-372. (24) Stemmler, E. A.; Buchanan, M. V.; Hurst, G. B.; Hettich, R. L. Anal. Chem. 1995, 67, 2924-2930. (25) Tang, K.; Taranenko, N. I.; Allman, S. L.; Chen, C. H.; Chang, L. Y.; Jacobson, K. B. Rapid Commun. Mass Spectrom. 1994, 8, 673-678. (26) Lecchi, P.; Le, H. M. T.; Pannell, L. K. Nucleic Acids Res. 1995, 23, 12761277. (27) Pieles, U.; Zu ¨ rcher, W.; Scha¨r, M.; Moser, H. E. Nucleic Acids Res. 1993, 21, 3191-3196. (28) Wu, K. J.; Stedding, A.; Becker, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 191. (29) Currie, G. J.; Yates, J. R., III J. Am. Soc. Mass Spectrom. 1993, 4, 955-963. (30) Liu, Y. H.; Bai, J.; Zhu, Y.; Liang, X.; Siemieniak, D.; Venta, P. J.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 735-743. (31) Tang, K.; Allman, S. L.; Chen, C. H. W.; Chang, L. Y.; Schell, M. Rapid Commun. Mass Spectrom. 1994, 8, 183-186. (32) Doktycz, M. J.; Hurst, G. B.; Habibi-Goudarzi, S.; McLuckey, S. A.; Tang, K.; Chen, C. H.; Uziel, M.; Jacobson, K. B.; Woychik, R. P.; Buchanan, M. V. Anal. Biochem. 1995, 230, 205-214. (33) Bai, J.; Liu, Y. H.; Liang, X.; Zhu, Y.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 1172-1176.
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responsible for this mass range limitation.40-45 Electrospray ionization (ESI) mass spectrometry offers a more gentle desorption process which allows analysis of intact DNA molecular ions.46-57 In this technique, multiply charged molecular ions are generated, and the mass spectra obtained are characterized by a distribution of peaks, where each peak represents an individual charge state of the molecule.58 The number of possible charge states increases with the size of the analyte, yielding fairly complex mass spectra for large molecules. The analysis of biopolymer mixtures by ESI mass spectrometry is, therefore, limited by the complexity of the overlapping peak distributions produced. Gel permeation chromatography (GPC)59-62 allows separation of polymers according to their external shape and dimensions. Although nucleic acids have been successfully analyzed by GPC,62,63 long separation times and poor column-to-column reproducibilities compromise the routine applicability of this technique. A variety of particle sizing methodologies are also being considered as potential detection techniques for macromolecules. DNA analysis by light scattering in free solution has been investigated and may prove a feasible approach in the future.64-66 (34) Chang, L. Y.; Tang, K.; Schell, M.; Ringelberg, C.; Matteson, K. J.; Allman, S. L.; Chen, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 772-774. (35) Tang, K.; Taranenko, N. I.; Allman, S. L.; Chang, L. Y.; Chen, C. H. W. Rapid Commun. Mass Spectrom. 1994, 8, 727-730. (36) Juhasz, P.; Roskey, M. T.; Smirnov, I. P.; Haff, L. A.; Vestal, M. L.; Martin, S. A. Anal. Chem. 1996, 68, 941-946. (37) Shaler, T. A.; Tan, Y.; Wickham, J. N.; Wu, K. J.; Becker, C. H. Rapid. Commun. Mass Spectrom. 1995, 9, 942-947. (38) Roskey, M. T.; Sminov, I. P.; Juhasz, P.; Vestal, M.; Talkach, E. J.; Martin, S. A.; Haff, L. A. Gen. Sci. Technol. 1995, 1, 46. (39) Kirpekar, F.; Nordhoff, E.; Kristiansen, K.; Roepstorff, P.; Hahner, S.; Hillenkamp, F. Rapid Commun. Mass Spectrom. 1995, 9, 525-531. (40) Zhu, L.; Parr, G. R.; Fitzgerald, M. C.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1995, 117, 6048-6056. (41) Tang, W.; Zhu, L.; Nelson, C. M.; Smith, L. M. Submitted for publication. (42) Hettich, R. L.; Buchanan, M. V. J. Am. Soc. Mass Spectrom. 1991, 2, 2230. (43) Kirpekar, F.; Nordhoff, E.; Kristiansen, K.; Roepstorff, P.; Lezius, A.; Hahner, S.; Karas, M.; Hillenkamp, F. Nucleic Acids Res. 1994, 22, 3866-3870. (44) Nordhoff, E.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Lezius, A.; Kirpekar, F.; Kristiansen, K.; Muth, J.; Meier, C.; Engels, J. W. J. Mass Spectrom. 1995, 30, 99-109. (45) Stemmler, E. A.; Hettich, R. L.; Hurst, G. B.; Buchanan, M. V. Rapid Commun. Mass Spectrom. 1993, 7, 828-836. (46) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451-4471. (47) Voress, L. Anal. Chem. 1994, 66, 481A-486A. (48) Smith, R. D.; Loo, J. A.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991, 10, 359-451. (49) Smith, R. D.; Loo, J. A.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (50) Bruins, A. P. J. Chem. Phys. 1993, 90, 1335-1344. (51) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990, 62, 882-899. (52) Valaskovic, G. A.; Kelleher, N. L.; Little, D. P.; Aaserud, D. J.; McLafferty, F. W. Anal. Chem. 1995, 67, 3802-3805. (53) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (54) Bleicher, K.; Bayer, E. Biol. Mass. Spectrom. 1994, 23, 320-322. (55) Bayer, E.; Maier, M.; Gaus, H. Anal. Chem. 1994, 66, 3858-3863. (56) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. Rapid Commun. Mass Spectrom. 1988, 2, 249-256. (57) Potier, N.; Dorsselaer, A. V.; Cordier, Y.; Bischof, R. Nucleic Acids Res. 1994, 22, 3895-3903. (58) Smith, E. L.; Alves, C. A.; Anderegg, J. W.; Porter, M. D.; Siperko, L. M. Langmuir 1992, 8, 2707-2713. (59) Barth, H. G.; Boyes, B. E.; Jackson, C. Anal. Chem. 1994, 66, 595R-620R. (60) Bly, D. L. Science 1970, 168, 527-533. (61) Yau, W. W.; Kirkland, J. J.; Bly, D. D. Modern Size Exclusion Liquid Chromatography; Wiley, New York, 1979. (62) Potschka, M. Anal. Biochem. 1987, 162, 47-64. (63) Kato, Y.; Sasaki, M.; Hashimoto, T.; Murotsu, T.; Fukushige, S.; Matsubara, K. J. Chromatogr. 1983, 266, 341-349. (64) Ware, B. R.; Flygare, W. H. J. Colloid Interface Sci. 1972, 39, 670-675.
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Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
Recently, it was demonstrated that biopolymers such as globular proteins could be analyzed using an electrospray scanning mobility particle sizer (ES-SMPS).66 In this technique, the macromolecule analyte solution is sprayed into fine, highly charged droplets in the electrospray source. The droplet charge is subsequently reduced in a bipolar neutralizer, and singly charged macromolecules are size-separated according to their mobility in air.66-71 We report here the first example of single- and double-stranded DNA molecule analysis using a scanning mobility particle sizer. The molecule sizes ranged from 6.1 kDa (20 nucleotides singlestranded DNA) to 300 kDa (500 base-pair double-stranded DNA), and as little as a few femtomoles was required to detect an analyte signal. Separation and detection were performed in about 3 min for each analyte. EXPERIMENTAL SECTION Aqueous ammonium acetate buffer was prepared (∼20 mM) with ultrapure water using reagent-grade ammonium acetate (Aldrich, Milwaukee, WI). The pH was 6.8 for the data shown in Figures 2, 3, 6-10, while it was 7.8 for the data of Figure 4. The buffer was degassed in a vacuum bell jar for about 1 h. The DNA oligonucleotides used in this work were d(ACGT)5 (20-mer, 6.1 kDa), dCTA GTG GCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT GGC GT (53-mer, 16.3 kDa), dT50 (50-mer, 15.1 kDa), and dT30AT80 (111-mer, 33.7 kDa). They were made by the University of Wisconsin Biotechnology Center on a DNA synthesizer Model 394 (ABI-Perkin Elmer, Foster City, CA) and purified by passage over a C18 SEP-PAK cartridge (Waters, Milford, MA). The 200 and 500 base-pair double-stranded DNA molecules were synthesized using a GeneAmp PCR kit (Perkin Elmer, Palo Alto, CA). λ DNA template, primers, enzyme, and reagents were used as supplied in the kit, and the PCR cycling parameters were set according to the manufacturer’s protocol. These double-stranded molecules were purified using a QIAquick PCR purification kit (Qiagen Inc., Chatsworth, CA). All the analytes were dissolved in the ammonium acetate buffer at a concentration of 50 µg/mL. Further dilutions from these stock solutions were made into the buffer just prior to the measurements. The electrospray scanning mobility particle sizer used in this work was described in detail previously (Figure 1).66,72 Briefly, the liquid was sprayed in the electrospray source directly from a fused silica capillary (Polymicro Technologies, Phoenix, AZ; inside diameter 25 µm, outside diameter 150 µm). This capillary was held by a grounded support needle and mounted concentrically in a sheath-gas tube. The inlet of the capillary was immersed in the sample vial, and the analyte solution flowed into the capillary due to the reduced pressure in the analyzer system. The pressure difference was fixed, and the flow rate was estimated at 50 nL/ min by timing the transit of a bubble through the capillary. A grounded Pt wire was immersed in the sample and maintained at ground potential. To avoid corona discharge,73 filtered CO2 was fed through the sheath tube at 0.1 L/min. The capillary was (65) Lee, M. R.; Smith, L. M. Unpublished results. (66) Kaufman, S. L.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Lewis, K. C. Anal. Chem. 1996, 68, 1895-1904. (67) Liu, B. Y. H.; Pui, D. Y. H. J. Colloid Interface Sci. 1974, 49, 305-312. (68) Whitby, K. T.; Liu, B. Y. H. Atmos. Environ. 1968, 2, 103-116. (69) Wang, S. C.; Flagan, R. C. Aerosol Sci. Technol. 1990, 13, 230-240. (70) Knutson, E. O.; Whitby, K. T. J. Aerosol Sci. 1975, 6, 443-451. (71) Zarrin, F.; Kaufman, S. L.; Socha, J. R. J. Aerosol Sci. 1991, 22, 343-346. (72) Chen, D.; Pui, D. Y. H.; Kaufman, S. L. J. Aerosol Sci. 1995, 26, 963-977. (73) Zeleny, J. Proc. Cambridge Philos. Soc. 1915, 18, 71.
Figure 2. Electrophoretic mobility diameter (EMD) spectrum of the oligonucleotide d(ACGT)5 (6.1 kDa). The analyte concentration was 1.25 µg/mL (0.2 pmol/µL). This spectrum is an average of four runs. A total of 0.12 pmol was used to record the spectrum.
Figure 1. System interconnections, showing volume flow rates. The DMA has aerosol inlet and outlet ports and also a closed “sheath loop” in which 20 L/min recirculates through a filter. This modified DMA also uses a 10 cm3/min purge flow of CO2 to prevent tailing due to aerosol recirculation in the outlet port. The vacuum/pressure regulator is set to keep the system pressure at 50 in. of water below atmospheric pressure. The exhaust port of the CPC is connected back to the vacuum regulator through a filter so that the CPC’s internal pump does not have to overcome this pressure difference. HV1 is the electrospray supply, adjusted to about 2.5 kV, and HV2 is the DMA supply, which is scanned up to around 5 kV. For other parameters, see the Experimental Section (Adapted from Figure 1 of ref 66).
positioned in the spraying chamber 3 mm from a 3 mm diameter orifice opening into the neutralization chamber. Filtered air was used as bath gas and supplied from the back of the spray chamber at 2 L/min. The orifice and neutralizing chamber were held at a potential of -2.5 kV. The air flowed through the orifice of the neutralizing chamber, sweeping the sprayed droplets into the chamber. A 210Po R source (NRD, Inc., Grand Island, NY) of about 1 mCi produces ionization sufficient to reach the Fuchs steadystate charge distribution inside the chamber, as shown by Liu and Pui.66,67,74,75 Under these steady-state conditions, multiply charged species entering this region lose their charge rapidly, to yield mostly neutral and singly charged species. The expected charge distribution has been calculated by Wiedensohler.75 The aerosol exited the neutralizing chamber through an outlet and was transported via conductive polymer tubing to the mobility analyzer. The differential mobility analyzer (DMA) used in this work was a modified Model 3071 (TSI, Inc., St Paul, MN).66 The axial air flow in the cylindrical mobility analyzer was 20 L/min. A high voltage (up to 5 kV) was applied to the center rod to create an electric field between the rod and the grounded cylinder wall. The voltage was adjusted stepwise to collect most airborne particles on the center rod, while gas phase macromolecules of a specific electrophoretic mobility Z could travel freely through the analyzer. The selected macromolecules were detected in a condensation particle counter (CPC) Model 3025 (TSI, Inc.),66,72,76,77 (74) Hinds, W. C. Aerosol Technology; Wiley: New York, 1982; p 304. (75) Wiedensohler, A. J. Aerosol Sci. 1988, 19, 387-389. (76) Lewis, K. C.; Dohmeier, D. M.; Jorgenson, J. W.; Kaufman, S. L.; Zarrin, F.; Dorman, F. D. Anal. Chem. 1994, 66, 2285-2292. (77) Wang, A. P. L.; Guo, X.; Li, L. Anal. Chem. 1994, 66, 3664-3675.
with butanol as the saturating vapor. The temperatures used for the saturator and the condenser were 37 and 10 °C, respectively. The data were acquired using commercial software (TSI SMPS version 2.0, Model 390089). The electrophoretic mobility Z of the molecules was measured, and their corresponding apparent electrophoretic mobility diameter (EMD) was obtained by inverting the Millikan formula:66,76,77
Z(EMD) ) (e/3πηEMD) × {1 + (λ/EMD)[2.514 + 0.8 exp(0.55(EMD/λ))]} (1) The gas viscosity η was assumed to be 1.834 × 105 kg m-1 s-1, and the molecular mean free path, λ, was taken to be 76.6 nm at the operating pressure. Data points were connected using a spreadsheet built-in interpolation routine (Cricket Graph, Computer Associates International, Inc., Islandia, NY). RESULTS AND DISCUSSION This study was undertaken to evaluate the performance of ESSMPS as a DNA analysis technique. The shortest DNA molecule tested was the 20-mer mixed-base oligonucleotide d(ACGT)5 (6.1 kDa). Figure 2 shows the electrophoretic mobility diameter (EMD) spectrum obtained for this compound. A peak is obtained corresponding to a diameter of 3.55 nm. The signal evident below 3 nm was attributed to aggregated nonvolatile impurities. A simple spherical model was used to provide a first-order interpretation of the observed EMDs. In this model, the EMD is related to the mass m of the molecule through the density equation:
density (d) ) mass/volume with mass ) m volume ) π(EMD)3/6 for a sphere of diameter EMD
The calculated EMDs were, therefore, obtained by rearranging the density equation, yielding 3
EMD )
x6m πd
Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
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Table 1. Measured and Calculated DNA Electrophoretic Mobility Diametersa EMD (nm) calcd DNA
moleculeb
ss20 bp monomer ss20 bp dimer ss20 bp trimer ss50 bp monomer ss50 bp dimer ss50 bp trimer ss53 bp monomer ss53 bp dimer ss53 bp trimer ss111 bp monomer ss111 bp dimer ss111 bp trimer ds200 bp monomer ds200 bp dimer ds200 bp trimer ds500 bp monomer ds500 bp dimer ds500 bp trimer
mass (kDa)
exptl
d ) 0.95
d ) 0.7
6.1 12.2 18.3 15.1 30.2 45.3 16.3 32.6 48.9 33.7 67.4 100.1 120 240 360 300 600 900
3.55 shoulder shoulder 3.71 4.66 shoulder 3.9 4.8
2.73 3.44 3.94 3.69 4.65 5.33 3.8 4.77 5.47 4.83 6.08 6.94 7.37 9.29 10.63 10.01 12.61 14.43
3.02 3.81 4.36 4.09 5.15 5.90 4.2 5.29 6.05 5.35 6.73 7.68 8.16 10.28 11.77 11.06 13.96 15.98
4.81 shoulder 8.1 11
a The density d is in g/cm3. EMDs were calculated for the dimer and trimer of each molecule. The experimental EMDs for multimers were given when readily identifiable on the spectra. Shoulders were also indicated when observed on the spectra. b ss, single-stranded, ds, double stranded.
Figure 3. EMD spectrum of the 53-mer oligonucleotide (16.3 kDa, see Experimental Section for sequence). The shoulder right of the main peak is attributed to dimer formation (see text). The concentration was 2.5 µg/mL (0.16 pmol/µL). This spectrum is an average of four runs. A total of 0.1 pmol was used to record the spectrum.
Table 1 shows the calculated EMDs for all the DNA molecules analyzed in this work, using the two density values found to best fit the data obtained for single-stranded and double-stranded DNAs, respectively (see below), as well as the EMDs measured by SMPS. The EMD spectrum for the 53-mer mixed-base oligonucleotide (16.3 kDa) is shown in Figure 3 (see previous section for sequence). The major peak of this spectrum corresponds to a diameter of 3.9 nm, while a shoulder peak is observed for D ≈4.8 nm. This secondary peak is attributed to formation of a dimer analyte. Multimers occur when several molecules are contained in a single electrospray droplet.66,74,76 Figure 4 further illustrates this effect for the analyte oligonucleotide dT50 (15.1 kDa). Figure 4A shows the EMD spectrum obtained at a concentration of 5 µg/mL (0.33 pmol/µL). The monomer peak is observed at 3.71 nm, while a fairly intense dimer peak is obtained at 4.66 nm. The amplitude ratio for these two peaks is about 0.58. Figure 4B,C shows the EMD spectra for the same 922 Analytical Chemistry, Vol. 69, No. 5, March 1, 1997
Figure 4. EMD spectra of dT50 (15.1 kDa). All the spectra are averages of four runs. (A) Concentration of 5 µg/mL (0.32 pmol/µL), 0.2 pmol used to record the spectrum; the secondary peak is attributed to the dimer. (B) Concentration of 2.5 µg/mL (0.16 pmol/ µL), 0.1 pmol used to record the spectrum. (C) Concentration of 1.25 µg/mL (0.08 pmol/µL), 0.05 pmol used to record the spectrum.
analyte as the concentration is reduced to 2.5 (0.16 pmol/µL) and 1.25 µg/mL (0.08 pmol/µL), respectively. The corresponding major/minor peak amplitude ratios are 0.26 and 0.15. This means that the secondary peak intensity decreases at lower concentration, consistent with dimer formation. Dimers can be formed before the electrospray process, through association-equilibrium in solution, as well as during the electrospraying, when two free molecules are captured in the same spray droplet.66 In the particular case of the polythymidine dT50, solution association is less likely because this analyte does not offer any base-pair selfcomplementarity for hybridization. The peak area for the monomer 50-mer was measured at three different concentrations to evaluate the potential for quantitation. Figure 5 shows the peak area versus concentration plot. The linear regression line is
Figure 5. Plot of the 50-mer monomer peak area versus concentration for the EMD spectra shown in Figure 4. The solid line is the regression line for these data (r ) 0.999).
Figure 7. EMDs plotted against oligonucleotides molecular weights. O, 20-mer single-stranded DNA; b. other single-stranded molecules; 0, double-stranded molecules. The solid and dashed lines show EMDs calculated using eq 2 and density values of 0.95 and 0.7 g/cm3, respectively.
Figure 6. EMD spectrum of dT30AT80 (33.7 kDa). The concentration was 3 µg/mL (90 fmol/µL). This spectrum is an average of four runs. A total of 50 fmol was used to record the spectrum.
plotted as well, and the regression coefficient is r ) 0.999. As in previous work with proteins, a linear relationship is, therefore, obtained between analyte concentration and peak area.66 A larger single-stranded oligonucleotide was specifically designed to test the possibility of fragmentation of the analyte during analysis. Fragmentation of oligonucleotides has not been observed in routine electrospray mass spectrometry.51-57 However, in MALDI mass spectrometry, it was found that oligonucleotides fragment preferentially at A, C, and G sites, while the T sites remain stable.39-44 The gas phase fragmentation mechanism is thought to be similar to the solution DNA hydrolysis process elucidated in the 1960s and 1970s.78-80 The sequence dT30AT80 was thus synthesized as a test compound to look for DNA fragmentation; cleavage at the A 3′ carbon-oxygen bond would yield two well-resolved fragments, dT30A and dT80. Figure 6 shows the spectrum obtained for this 111-mer oligonucleotide. Only one peak is obtained, and no evidence of fragmentation was detected for the other mixed-base oligonucleotides used in this study, suggesting that DNA fragmentation does not occur in this technique. Interestingly, old oligonucleotide samples were also analyzed by ES-SMPS, and multiple peaks were visible on the EMD spectra (data not shown), suggesting that the samples may have degraded. A second analysis using MALDI mass spectrometry confirmed that these samples were, in fact, degraded before analysis. Comparison of the observed and calculated EMDs for the oligonucleotides in Table 1 shows a reasonable agreement for (78) Kochetkow, H. K.; Budovskii, E. I. Organic Chemistry of Nucleic Acids; Plenum Publishing Co. Ltd.: London, 1972. (79) Brown, D. M.; Todd, A. R. Nucleic Acids; Academic Press: London, 1955; Vol. 1. (80) Michelson, A. M. The Chemistry of Nucleosides and Nucleotides; Academic Press: London, 1963.
single-stranded DNA when d ) 0.95 g/cm3. Figure 7 shows a plot of measured EMD versus analyte mass. EMDs calculated with eq 2 and using a value for d of 0.95 g/cm3 are also shown (dashed line). The main experimental deviation from this line is observed for the 20-mer oligonucleotide. Since the detection of the CPC drops abruptly below about 3 nm, it becomes difficult to make reliable EMD measurements for molecules near this cutoff. The deviation obtained here for the 20-mer oligonucleotide (EMD ) 3.55 nm) is consistent with a similar deviation observed for insulin in the protein study.81 This may be due to the proximity of the cutoff, or, alternatively, it could be due to a shell of nonvolatile small-molecule solutes, bound solvent molecules, or a combination of both. Small diameter species which retain a shell would have their EMD affected in greater proportion than species of larger diameter, assuming either constant shell thickness (expected for bound solvent) or constant shell mass (as would occur with nonvolatile solutes). This would, therefore, explain the greater deviation observed for the small-diameter species on the EMD versus mass plot. Figure 8A,B shows the EMD spectra obtained for the 200mer double-stranded DNA molecule (120 kDa) at 3 and 1 µg/ mL, respectively. As noted before, the signal recorded at low diameter values is attributed to nonvolatile impurities. This signal is more noticeable in these spectra due to the lower signal/noise ratio. In Figure 8A, a high concentration of aggregated impurities is observed at a diameter of about 4 nm. When the sample is diluted 3-fold (Figure 8B), this phenomenon is reduced, and the aggregates are no longer apparent. The EMD spectrum for the 500-mer double-stranded DNA (300 kDa) is shown in Figure 9. Table 1 shows that a density of 0.7 g/cm3 provides reasonable agreement between the experimental and calculated EMD values for double-stranded DNA (see Figure 7). This density is substantially lower than the value determined for the single-stranded molecules (d ) 0.95 g/cm3). This difference could be explained on the basis of the greater rigidity of double-stranded DNA molecules. The persistence length of double-stranded DNA in solution was previously estimated at about 200 nucleotides, while it is expected to be of the order of a few nucleotides for single(81) Kaufman, S. L.; Skogen, J. W.; Dorman, F. D.; Zarrin, F.; Lewis, K. C. Correction submitted to Anal. Chem.
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Figure 10. EMD spectrum of a quaternary mixture containing (a) dT50 (0.8 µg/mL, 50 fmol/µL), (b) dT30AT80 (1.5 µg/mL, 45 fmol/µL), (c) double-stranded 200-mer (2.5 µg/mL, 21 fmol/µL) and (d) doublestranded 500-mer (4.0 µg/mL, 13 fmol/µL). The spectrum is an average of three runs, and 450 nL was used for the spectrum.
Figure 8. EMD spectra of the 200-mer double-stranded DNA (120 kDa). The spectra shown are four run averages. (A) Concentration of 3 µg/mL (25 fmol/µL), a total of 15 fmol used for the spectrum. (B) Concentration was 1 µg/mL (8.3 fmol/µL), a total of 5 fmol used for the spectrum.
Figure 9. EMD spectra of the 500-mer double-stranded DNA (300 kDa). This spectrum is an average of four runs. The concentration was 1.5 µg/mL (5 fmol/µL), and a total of 3 fmol was used for the spectrum.
stranded molecules.82-86 Although these earlier studies were performed in solution phase, a comparable flexibility difference between double- and single-stranded DNA may also exist in the gas phase. In this model, the single-stranded molecules would fold more tightly than the double-stranded molecules, hence presenting a higher apparent density. However, these conclusions remain speculative at this point, as a difference in solvent and/or (82) Slater, G. W.; Lalande, M. Biopolymers 1988, 27, 509-524. (83) Rosenberg, A. H.; Studier, F. W. Biopolymers 1969, 7, 765-774. (84) Borochkov, N.; Eisenberg, H.; Kam, Z. Biopolymers 1981, 20, 231-235. (85) Holmes, D. L.; Stellwagen, N. C. Electrophoresis 1990, 11, 5-15. (86) Stellwagen, N. C. Biopolymers 1985, 24, 2243-2255.
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impurity aggregation propensity between single- and doublestranded DNA may also explain the observed density difference. The sensitivity of ES-SMPS to molecular conformation could be evaluated in future work by studying a single type of biopolymer in different temperature or pH conditions, as performed previously with a number of ESI-based methodologies.87-96 The ability of ES-SMPS to analyze mixtures was evaluated by analyzing a solution containing the 50-, 111-, 200-, and 500-mers (Figure 10). Peak width and resolution of the DMA have been studied in detail previously.66 From this study and the results of Figure 10, we estimate that a difference of about 30% in molecular weight is required in order to achieve separation between species at this point. It is noteworthy that results presented in this work were obtained without major modification of the SMPS hardware. We anticipate that better separation power will be achieved by optimizing the instrument design and flow conditions. CONCLUSIONS We demonstrated that an electrospray scanning mobility particle sizer can be used to analyze single- and double-stranded DNA molecules. The samples used in this study ranged from 6.1 to 300 kDa, and separation and detection were performed at picomole and femtomole levels in a few minutes. No fragmentation of the DNA analytes was detected. A simple spherical model was used to correlate the measured electrophoretic mobility diameters with molecular weights. In addition, the data indicated an effective density difference between single-stranded and doublestranded DNA molecules. We anticipate that optimization of running parameters as well as instrument hardware will make this (87) Clemmer, D. E.; Hudgins, R. J.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141-10142. (88) Shumate, C. B.; Hill, H. H. Anal. Chem. 1989, 61, 601-606. (89) Wittmer, D.; Chen, Y. H.; Luckenbill, B.; Hill, H. H. Anal. Chem. 1994, 66, 2348-2355. (90) Cox, K. A.; Julian, R. K.; Cooks, R. G.; Kaiser, R. E. J. Am. Soc. Mass Spectrom. 1994, 5, 127-128. (91) Feng, R.; Konishi, Y. J. Am. Soc. Mass. Spectrom. 1993, 4, 638-645. (92) LeBlanc, J. C. Y.; Beuchemin, D.; Siu, K. W. M.; Guevremont, R.; Berman, S. S. Org. Mass Spectrom. 1991, 26, 831-839. (93) Guevremont, R.; Siu, K. W. M.; LeBlanc, J. C. Y.; Berman, S. S. J. Am. Soc. Mass Spectrom. 1992, 3, 216-224. (94) Smith, R. D.; Light-Wahl, K. J. Biol. Mass Spectrom. 1993, 22, 493-501. (95) Loo, R. R. O.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1994, 5, 207-220. (96) Loo, J. A.; Ogorzalek, R. R.; Udseth, H. R.; Edmons, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101-105.
technique a fast and sensitive analysis method for separation of DNA and other biopolymers. In addition, the charge reduction process utilized in ES-SMPS is expected to be useful to avoid multiple charging in ESI-MS, in particular when used in conjunction with a time-of-flight (TOF) analyzer. The feasibility of this approach is being investigated in our laboratory. ACKNOWLEDGMENT We thank Dr. Meng-Rong Lee for her assistance with the preparation of double-stranded DNA. This work was supported
by Department of Energy Human Genome Grant DEFG0291ER61130 and by TSI Inc.
Received for review August 2, 1996. Accepted December 3, 1996.X AC960785K
X
Abstract published in Advance ACS Abstracts, January 15, 1997.
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