Characterization of DNA Oligonucleotides by Coupling of Capillary

limit for the 5′-monophosphate nucleotides dAMP and. TMP was 30 fmol injected for single MS and 400 fmol for a collision-induced daughter ion spectr...
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Anal. Chem. 1998, 70, 3060-3068

Characterization of DNA Oligonucleotides by Coupling of Capillary Zone Electrophoresis to Electrospray Ionization Q-TOF Mass Spectrometry Dieter L. D. Deforce,† Jos Raymackers,‡ Lydie Meheus,‡ Frans Van Wijnendaele,‡ Andreas De Leenheer,† and Elfriede G. Van den Eeckhout*,†

Laboratory for Pharmaceutical Biotechnology, University of Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium, and Innogenetics, Industriepark Zwijnaarde 7 Box 4, B-9052 Ghent, Belgium

A procedure for fast and precise molecular weight, purity, and base composition determination of oligonucleotides (in the size range of 20-120 bases) using capillary zone electrophoresis (CZE) coupled by an electrospray interface (ES) on-line to a quadrupole-inlet time-of-flight mass spectrometer (Q-TOFMS) is described. The detection limit for the 5′-monophosphate nucleotides dAMP and TMP was 30 fmol injected for single MS and 400 fmol for a collision-induced daughter ion spectrum (CID-MS/ MS). Injection of 1 pmol was the detection limit for the oligonucleotides. The use of the CZE as an injection interface for oligonucleotides successfully removed metal ion adducts, greatly enhancing the sensitivity and the precision of the mass detection without the need for sample preparation steps. Using electrophoretic separation in an ammonium carbonate buffer system (0.1 M, pH 9.68), these metal adducts were removed and exchanged for volatile ammonium ions. A simple sample stacking technique was developed in order to be able to inject sufficient amounts of sample on the capillary prior to electrophoresis. Using this sample stacking technique, the oligonucleotides could be concentrated up to a factor of 100 on the capillary. Given the high mass precision ((50 ppm) and the high mass resolution (peak widths at half-height of 4 Da), CZE-Q-TOFMS proves to be very useful not only for determination of the purity and the length of oligonucleotides (up to 120 bases) but also for confirmation of the expected base composition, making this technique an extremely useful tool for quality control in the field of oligonucleotides.

methods used for this purpose were gel electrophoresis, capillary gel electrophoresis (CGE), and high-performance liquid chromatography (HPLC). These methods are based on the separation of the oligonucleotides according to their length, regardless of their base sequence or composition, and are thus not sufficient for absolute identification. Recently, a variety of mass spectral techniques were developed to characterize oligonucleotides. Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) has proven to be able to size oligonucleotides1-3 up to a length of about 260 bases;4 however, some drawbacks to the use of MALDI are the need for a tedious sample preparation and, more importantly, the low mass resolution and sensitivity for oligonucleotides more than 50 base pairs (bp) in length.4,5 In particular, the low mass resolution has made this technique, until now, not suitable for identification and characterization of oligonucleotide probes,6 which are up to 120 bases in length. Another approach is the use of ES ionization techniques. Until recently, this ionization technique was used in combination with quadrupole mass analyzers for the identification of oligonucleotides.7-9 ES ionization allows on-line coupling of a separation technique such as HPLC7 or CZE.10 However, the low mass resolution of quadrupole instruments makes them useful only for length determination of oligonucleotides with a length of up to 80 bases.8,9 The use of ES with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR), which has a superior mass resolution, enabled the identification and characterization of oligonucleotides up to 120 bases;6,11,12 however, this is not a commonly available instrument.

* To whom correspondence should be addressed. Tel. +32 9 221 99 43. Fax: +32 9 220 66 88. E-mail: [email protected]. † University of Ghent. ‡ Innogenetics.

(1) Ball, W. R.; Packman, L. C. Anal. Biochem. 1997, 246, 185-194. (2) Little, D. P.; Cornish, T. J.; O’Donnell, M. J.; Braun, A.; Cotter, R. J.; Ko ¨ster, H. Anal. Chem. 1997, 69, 4540-4546. (3) Wei, J.; Lee, C. S. Anal. Chem. 1997, 69, 4899-4909. (4) Ross, P. L.; Belgrader, P. Anal. Chem. 1997, 69, 3966-3972. (5) Jurinke, C.; van den Boom, D.; Collazo, V.; Lu ¨ chow, A.; Jacob, A.; Ko ¨ster, H. Anal. Chem. 1997, 69, 904-910. (6) Muddiman, D. C.; Wunschel, D. S.; Liu, C.; Pasa-Tolic, L.; Fox, K. F.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 3705-3712. (7) Apffel, A.; Chakel, J. A.; Fisher, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325. (8) Limbach, P. A.; Crain, P. F.; McCloskey, J. A. J. Am. Soc. Mass Spectrom. 1995, 6, 27-39. (9) Potier, N.; Van Dorsselaer, A.; Cordier, Y.; Roch, O.; Bischoff, R. Nucleic Acids Res. 1994, 22, 3895-3903. (10) Deforce, D. L. D.; Ryniers, F. P. K.; Lemie`re, F.; Esmans, E. L.; Van den Eeckhout, E. G. Anal. Chem. 1996, 68, 3575-3584.

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S0003-2700(98)00147-4 CCC: $15.00

PCR technology and the use of probe hybridization techniques for the detection of these PCR products has become a very important tool in the field of DNA analysis in biology, microbiology, medicine, and forensics. Especially in the fields of medicaldiagnostics and forensics, quality control and characterization of the PCR primers and probes are of major importance to give the results the needed degrees of certainty. Until now, the analytical

© 1998 American Chemical Society Published on Web 06/18/1998

Since the mass spectrometric determination greatly benefits from the removal of metal ion adducts (for both MALDI-TOFMS and ES-FTICR), a variety of sample pretreatment schemes were developed to remove metal ions. In general, these procedures involve repeated ethanol precipitations in the presence of ammonium acetate,8,13,14 the use of commercial purification kits,15,16 magnetic beads-based purification,4,5 addition of chemicals such as trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid (CDTA) and triethylamine (TEA),8 addition of organic bases such as imidazole and piperidine,17 or on-line microdialysis.6 The goal of this paper is to investigate whether the new Q-TOFMS instrumentation, with significantly improved mass resolution over quadrupole instrumentation,18 can be used for the identification and characterization of oligonucleotides of up to 120 bases. Furthermore, the use of an on-line CZE-ES-Q-TOFMS separation is investigated for its ability to enhance the detection and characterization of oligonucleotides by the exchange of metal ions for ammonium ions during separation in the ammonium carbonate buffer, thus eliminating the need for any sample preparation steps. An easy-to-implement sample stacking technique, compatible with ES, was developed, allowing on-line concentration of the oligonucleotides on the capillary. The sample stacking technique described here does not require the use of makeup solvent switching as reported before,10 thus simplifying the procedure and shortening the overall analysis time. The described CZE separation offers the potential to separate PCR products from primers, salts, enzyme, and nucleotide precursors prior to MS analysis. EXPERIMENTAL SECTION Instrumentation. CZE separations were performed on a PRINCE Lauerlabs (Emmen, The Netherlands) system equipped for on-column detection with a Kontron UV detector (type HPLC 332) at a wavelength of 270 nm. Data collection was performed with the PC Integration Pack version 3.9 (Kontron Instruments, Milan, Italy). ES-Q-TOFMS was done on a Micromass (Manchester, UK) Q-TOF system equipped with a Mass Lynx data system. It is important to mention that this is a reflectron type of TOF, since this reflectron technology greatly enhances the mass resolution of the TOF. A triaxial ES probe adapted for CZE coupling was obtained from Micromass. Reagents. 2′-Deoxyadenosine-5′-monophosphate (dAMP) and thymidine-5′-monophosphate (TMP) were obtained from Sigma (St. Louis, MO). The short oligonucleotides (18-27-mers) were all synthesized by SGS (Copenhagen, Sweden), except for R-7081, R-6963, and R-6841, which were synthesized by Pharmacia (11) Muddiman, D. C.; Anderson, G. A.; Hofstadler, S. A.; Smith, R. D. Anal. Chem. 1997, 69, 1543-1549. (12) Aaserud, D. J.; Kelleher, N. L.; Little, D. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1996, 7, 1266-1269. (13) 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. (14) Hurst, G. B.; Doktycz, M. J.; Vass, A. A.; Buchanan, M. V. Rapid Commun. Mass Spectrom. 1996, 10, 377-382. (15) Liu, Y. H.; Bai, J.; Liang, X.; Lubman, D. M.; Venta, P. J. Anal. Chem. 1995, 67, 3482-3490. (16) Liu, Y. H.; Bai, J.; Zhu, Y.; Liang, X.; Semeniak, D.; Venta, P. J.; Lubman, D. M. Rapid Commun. Mass Spectrom. 1995, 9, 735-743. (17) Greig, M.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97-102. (18) Morris, H. R.; Paxton, T.; Panico, M.; McDowell, R.; Dell, A. J. Protein Chem. 1997, 16, 469-479.

(Uppsala, Sweden); these oligonucleotides were all biotinylated at the 5′-end (Table 1). The oligonucleotide coded as R-8630 contains one A-G wobble and is thus, in fact, a mixture of two distinct oligonucleotides, and R8633 contains an A-C wobble and one T-C wobble, thus containing four different oligonucleotides. The long oligonucleotides all consisted of a 100 T repeat unit and a variable 15-20-mer oligonucleotide unit (both synthesized by SGS), which were chemically linked to each other to give the described length and composition. The synthesized oligonucleotides were not purified or desalted before the analysis described here. HPLC-grade water was obtained from an Elga Maxima ultrapure water treatment device. Other products and solvents were of analytical grade and were used without further purification. Sample Stacking. The samples were diluted (1/1) in a 10 mM ammonium carbonate buffer (pH 9.68) and loaded on the autosampler. Samples were then concentrated on the capillary using the sample stacking technique procedure described below in order to be able to inject sufficient amounts of analyte without introducing a large sample plug, which would render electrophoresis impossible. The normal stacking10 procedure for negative analytes involves switching of the makeup solvent, used to obtain a stable electrospray, to the buffer used to perform electrophoresis. This is necessary because solvent gets drawn into the capillary by the bulk electroosmotic flow (EOF) at the outlet end of the capillary. In an attempt to simplify this procedure and shorten the analysis time, this procedure was slightly altered to eliminate the need for makeup solvent switching. During sample stacking, the voltages in the ionization probe were turned off to prevent the negative potential at the probe tip to cause sample discrimination of the negatively charged oligonucleotides. The capillary was filled with the electrophoresis buffer (0.1 M ammonium carbonate, pH 9.68), and sample stacking was then performed by the simultaneous application of a pressure of 40 mbar and an electrophoretic potential of -8 kV during 1 min. The negative potential concentrated the oligonucleotides at the buffer interface. The pressure of 40 mbar was needed to compensate the EOF caused by the application of -8 kV. Since both forces try to cause an opposite flow of solvent, the net flow was minimized, and only a very small sample plug was introduced. CZE-ES-Q-TOFMS. To couple the CZE system to the ESQ-TOFMS system, a fused silica capillary of 1 m × 75 µm i.d. (365 µm o.d.) was used, which was inserted inside the triaxial ES probe. The CZE capillary was positioned so that, at the tip of the ES probe, the CZE capillary protruded 200 µm with reference to the liquid sheath tube. The liquid sheath tube itself protruded 500 µm with reference to the capillary that introduces the nebulizing gas. The anode reservoir and the tip of the electrospray probe were positioned at the same height in order to prevent additional hydrodynamic effects. The Lauerlab PRINCE instrument allowed monitoring of the current during electrophoresis even when coupled to MS by using the current control at the inlet electrode. This is a prerequisite for sample stacking as described here. A makeup flow of 80% 2-propanol, 15% water, and 5% 0.01 M ammonium carbonate buffer (pH 9.68) was introduced using the sheath capillary at a flow rate of 10 µL/min, controlled by using an auxiliary HPLC pump. This makeup flow is necessary to obtain stable ES conditions and, at the same time, ensures electrical contact at the CZE capillary. The flow rate of the N2 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

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Table 1. Oligonucleotides Used in This Study MM (Da) no.

code

base composition

base length

1

R8630A R8630B R7666 R8683 R8960 R8962 R8958 R8959 R7647 R8633A R8633B R8633C R8633D R8632 R7081b R6963b R8684 R8683 R6841b R7415 DR5004 DR5005 DR5006 DR5007 DR5008 DR5009 DR5010 DR5011 DR5012 DR5013

biotine-[G7A6C8T6] biotine-[G8A5C8T6] biotine-[G6A4C5T5] biotine-[G6A3C8T5] biotine-[G8A4C7T6] biotine-[G7A4C6T7] biotine-[G7A2C6T3] biotine-[G3A5C8T3] biotine-[G6A6C6T7] biotine-[G8A5C4T9] biotine-[G8A4C5T9] biotine-[G8A5C5T8] biotine-[G8A4C6T8] biotine-[G5A7C10T4] biotine-[G5A4C10T5] biotine-[G6A3C8T5] biotine-[G5A4C10T5] biotine-[G6A3C8T5] biotine-[G5A4C10T5] biotine-[G6A3C5T5] [G6A4C3T103] [G3A7C5T104] [G8A2C3T103] [G8A3C5T102] [G7A5C4T100] [G7A3C5T100] [G6A7C5T102] [G7A4C4T103] [G5A5C6T102] [G2A8C6T105]

27 27 20 22 25 24 18 19 25 26 26 26 26 26 24 22 24 22 24 19 116 119 116 118 116 115 120 118 118 121

2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

expected average

observed

errora

8 666.064 8 682.063 6 538.685 7 093.029 8 079.669 7 765.471 5 922.266 6 123.429 8 062.682 8 437.914 8 413.888 8 422.902 8 398.877 8 290.832 7 655.398 7 093.029 7 655.398 7 093.029 7 655.398 6 225.475 35 365.898 36 504.662 35 397.897 35 985.279 35 689.109 35 047.678 36 579.700 35 984.292 35 913.256 36 777.847

8 666.610 8 682.730 6 538.300 7 092.330 8 079.420 7 765.780 5 922.850 6 123.380 8 063.230 8 438.240 8 414.300 8 423.110 8 399.420 8 291.120 7 743.340 7 180.410 7 656.060 7 093.540 7 743.750 6 225.830 35 365.410 36 503.030 35 399.030 35 987.010 35 690.450 35 048.560 36 580.550 35 985.160 35 914.340 36 778.090

63.0 76.8 -58.9 -98.6 -30.8 39.7 98.6 -8.0 68.0 38.7 48.9 24.7 64.7 34.7 +87.9 Da +87.4 Da 86.5 72.0 +88.4 Da 57.0 -13.8 -44.7 32.0 48.1 37.6 25.2 23.2 24.1 30.2 6.6

a In ppm unless noted otherwise. b These oligonucleotides were synthesized by Pharmacia and exhibit an observed MM which is, on average, 87.9 Da higher than the expected average MM (See text).

bath gas was adjusted between 50 and 100 L/h. The flow rate of the N2 nebulizing gas was set at 30 L/h. During injection of the samples and during sample stacking, the voltages in the ionization probe were turned off to prevent the negative potential at the probe tip from causing sample discrimination during injection (in our case, the analytes of interest were negatively charged and were discriminated during injection). Normal injection was performed by applying pressure, typically 22 mbar during 0.2 min. Negative (-) ES ionization was performed using an ionization voltage of -3.7 kV at the probe tip. The cone voltage was set at 30 V, and the source temperature was kept constant at 75 °C. All CZEES-Q-TOFMS results were obtained using a 0.1 M ammonium carbonate buffer (pH 9.68) as the buffer system for CZE. Electrophoresis was performed by applying 5 kV for 2 min, 7 kV for 2 min, and 10 kV until the end of the analysis. During electrophoresis, a constant pressure of 60 mbar was applied in order to shorten the analysis time. This results in a loss of separation resolution, but this resolution is no longer important, given the specificity of the mass analyzer. A scan range of 5001600 Da was covered at a scan speed of 920 Da/s for the detection of the oligonucleotides. Low-energy collision-activated dissociation (CAD) product ion spectra were obtained for the [M - H]- ions. The collision gas (Ar) pressure was 3 × 10-3 mbar, and the collision energy was optimized for each component by introducing a sample plug through the capillary using pressure. The optimal collision energy varied between 20 and 35 eV, depending on the component. 3062 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

Mass Calculations. The expected average molecular masses (MMs) of all oligonucleotides were calculated using BioLynx Software, which is part of the MassLynx software package (Micromass) (Table 1). Electrospray of oligonucleotides produces an envelope of multiply charged ions, typically in the m/z range of 400-2000 Da. Using the MaxEnt algorithm (Micromass), deconvoluted spectra were calculated, displaying the observed masses of the uncharged molecules (Table 1). Possible base compositions were calculated using a spreadsheet written in our laboratory under Lotus 1-2-3. RESULTS AND DISCUSSION Sample Stacking. Using a normal injection without sample stacking, the maximum volume we could inject was calculated to be 22 nL (following an injection of 22 mbar during 0.2 min). For a sample with a concentration of 10 × 10-6 M, this would result in the injection of 0.22 pmol of oligonucleotide. Since the samples were dissolved in a solution with an electrolyte concentration much lower than that of the running buffer, injection of a larger plug leads to the production of air bubbles in the capillary when a separation voltage was applied. However, this enables the use of sample stacking, where it is a prerequisite that the conductivity of the sample solution is lower than that of the running buffer. The critical parameters and the efficiency of the sample stacking technique described here were evaluated using UV detection. The amount of analyte injected was evaluated by integration of the UV signals. For a given injection voltage of -8 kV, we evaluated

Figure 1. Schematic representation of the sample stacking procedure. The left side shows the triaxial probe tip, which is positioned in the ES source. The right side depicts the injection from the injection vial. (A) Optimal sample stacking conditions: the pressure of 40 mbar compensates the EOF, and there is no net flow of solvent. (B) The pressure is too low to compensate the EOF, causing makeup solvent to flow in the capillary, and analytes are pushed out of the capillary. (C) The pressure is too high, and a large sample plug gets introduced into the capillary.

the pressure needed to compensate the EOF caused by the electric potential (Figure 1) For this purpose, we monitored the current on the CZE instrument. When the pressure is too high, and sample solvent gets injected on the capillary, the current gradually decreases (Figure 1, panel C). If electrophoresis is started at this point, the large plug will lead to the formation of bubbles in the capillary. When this pressure is too low, the analytes are unable to get in the capillary due to the EOF (Figure 1, panel B). When coupled to ES-Q-TOFMS, a low pressure would also cause makeup solvent to be drawn into the capillary, and since this solvent has a very low conductivity, this would also result in a current drop. The pressure was thus optimized at 40 mbar (Figure 1, panel A). Using these conditions for 1.2 min, 22 pmol of oligonucleotide could be injected from a sample with a concentration of 10 × 10-6 M. Compared to the normal injection, the sample was thus concentrated up to a factor of 100 on the capillary. Using these conditions, only a very small amount of sample solution was injected on the capillary, enabling subsequent electrophoretic separation. This method is readily amenable to automation since there is no need for makeup solvent switching. By monitoring the current generated during the injection, the pressure needed to compensate the EOF could be optimized

semiautomatically. The CZE instrument was programmed to decrease the pressure by 2 mbar when the current decreased by 1 µA. Starting with an initial pressure of 45 mbar, the CZE instrument automatically decreases the pressure to the level needed to compensate for the EOF (which was about 40 mbar). Detection Limits of CZE-ES-Q-TOFMS. The detection limits of the CZE-ES-Q-TOFMS system were determined using a stock solution of dAMP and TMP (10 mg of each were dissolved in 10 mL of HPLC-grade water, and further dilutions were also prepared in HPLC-grade water). This sample was chosen, although not representative for the analysis of oligonucleotides, to be able to evaluate the efficiency of this new combination of CZE to ES-Q-TOF in comparison to conventional quadrupole coupling of CZE.10 The detection limit in the single MS mode was determined to be 30 fmol (S/N 5:1) after normal injection (22 mbar, 0.2 min) of a sample with a concentration of 1.5 × 10-6 M and scanning a mass range from 50 to 800 Da (scan speed 1750 Da/s). This is a gain in sensitivity of about 20 times compared to that of a conventional quadrupole mass analyzer operating in full-scan mode (100-700 Da at a scan speed of 300 Da/s) for the same components.10 Using the sample stacking technique,10 a 200-fold increase in sensitivity could be obtained as compared to that of the normal injection, leading to a detection limit for CZE-ES-Q-TOFMS of 7.5 × 10-9 M. Using the same sample, the minimum amount of compound needed to obtain a CAD-induced daughter ion spectrum (collision energy was optimized at 21 eV) of TMP was determined to be 400 fmol injected on the CZE capillary. This amount is slightly lower than the detection limit in single MS in full-scan mode (100700 Da) on a conventional quadrupole mass analyzer.10 For the oligonucleotide R8958 (which is 18 bases long), spectra which could still be deconvoluted by the MaxEnt software were obtained from the injection of 1 pmol of oligonucleotide (out of a solution of 1 × 10-6 M) on the CZE-ES-Q-TOFMS, using the sample stacking technique described here. Analysis of the Long Oligonucleotides (120 Bases) Using CZE-ES-Q-TOFMS. All data were obtained by the injection of 10-40 pmol (sample solutions of 10-5-10-4 M) of oligonucleotide on the capillary using the sample staking technique. To investigate the influence of the electrophoretic separation on the metal ion exchange and on the sensitivity of the mass determination, oligonucleotide DR5004 was analyzed with and without electrophoresis after injection of 20 pmol. After the sample stacking procedure was performed, the sample plug was infused in the probe by applying a pressure of 60 mbar, without any electrophoretic potential, at the injection side of the CZE capillary. Using these conditions, only a very weak signal-to-noise envelope was obtained (Figure 2, panel A); it was impossible to obtain a deconvoluted spectrum. However, when the same sample was injected and an electrophoretic potential (for conditions, see Experimental Section) was applied during the transport of the sample plug through the capillary, the signal-to-noise ratio was greatly enhanced, and a nice envelope was obtained (Figure 2, panel B). This envelope was intense enough to obtain a deconvoluted spectrum (Figure 2, panel C) using Max Ent (for conditions, see Experimental Section). From this deconvoluted spectrum, it is obvious that the MM of 35 365.41 Da, corresponding with the mass of the expected product (35 365.89 Da), is not Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

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Figure 2. CZE-ES-Q-TOFMS mass spectra obtained for the long oligonucleotide DR5004. For both spectra, 20 pmol was injected on the CZE capillary (for conditions, see text). (A) Spectrum obtained without electrophoresis. (B) Spectrum obtained for the same sample using electrophoresis. (C) Deconvoluted spectrum of the raw spectrum in panel B. Deconvolution was performed using MaxEnt (see text). 3064 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

Figure 3. CZE-ES-Q-TOFMS mass spectrum obtained for the long oligonucleotide DR5008 (for conditions, see text). (A) Raw spectrum of the multiply charged ions of the oligonucleotide. The insets shows a detail of the mass range around the isotope cluster with a net charge of -37. (B) Deconvoluted spectrum obtained using MaxEnt.

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Figure 4. Deconvoluted mass spectrum obtained using MaxEnt for the analysis of R8958 (see text). Note the absence of any metal ion adducts. The inset shows the CZE-ES-Q-TOFMS electropherogram of the analysis of R8958. (A) Reconstructed ion electropherogram for the m/z value 1183.5 Da, being the ion with a net charge of -5. (B) Total ion electropherogram; notice the separation of the oligonucleotide (migration time 4.5-5.5 min) from the co-injected impurities (migration time 3.75-4.25 min).

the major component of this sample. Instead, a product with a MM of 134.5 Da less was the most abundant; this corresponds with the loss of one adenine base, probably due to depurination. Most of the long oligonucleotides (Figures 2 and 3) exhibit products with molecular mass 51 and 84 Da higher than the expected mass, probably resulting from the chemical linking of the oligonucleotide to the 100 T tail, as this was observed only in the spectra of the long oligonucleotides. Most of the spectra also revealed the presence of a product with a mass of 304 Da lower (Figure 3) than expected mass; this corresponds to the loss of one TMP residue, probably originating from the linking of a 99 T tail to the oligonucleotide. No sodium adducts were found in these deconvoluted spectra, indicating that the exchange for ammonium ions during the electrophoresis in the ammonium carbonate buffer system is very efficient. However, some of the spectra (Figure 3) reveal the presence of a magnesium adduct (+24 Da); this is in agreement with the findings of Limbach et al.,8 who reported that the adducts of divalent metals are more resistant to exchange with ammonium, during precipitation, than the monovalent cations. All these spectra were obtained within an analysis time of 10 min (Figure 4, inset). The oligonucleotides elute as a broad peak (migration time 4.5-5.5 min), due to the overloading of the capillary and the additional pressure used during electrophoresis; however, the loss in resolution is not significant compared to the information which is obtained by the mass analyzer. Note that 3066 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

the oligonucleotides are separated from the co-injected, nonoligonucleotide impurities (migration time 3.75-4.25 min). The use of the sample stacking technique with subsequent electrophoresis is a prerequisite to remove the metal ions from the oligonucleotide backbone, thus enhancing sensitivity and precision. Bearing in mind that these results are obtained without pretreatment of the sample and in a very short analysis time, this method is readily amenable for routine analysis of oligonucleotides. A comparison between observed and theoretical molecular masses obtained for all the investigated long oligonucleotides is given in Table 1. The peak width of an isotope cluster, with a net charge of -37, in the raw spectrum at half-height is 0.3 m/z unit (Figure 3, inset). This is approximately 10 times better than that achieved with a conventional quadrupole mass analyzer (2 m/z units).8 This leads to a reduced error in mass determination (0.004%) and to an enhanced resolution of the deconvoluted MaxEnt spectra (peak width at half-height in the deconvoluted spectra of 4 Da). This makes this technique extremely useful for identifying long oligonucleotides (up to 120 bases). All long oligonucleotides tested were readily identified, even though some of them have the same number of bases. It would thus have been impossible to identify these oligonucleotides by HPLC or gel electrophoretic techniques. A misincorporation of one basesin the worst case a T to A switchshaving a mass difference of 9 Da would be easily detected.

Figure 5. CZE-ES-Q-TOFMS mass spectrum obtained for the oligonucleotide with two wobbles, R8633 (for conditions, see text). (A) Raw spectrum of the multiply charged ions. The insets show a detail of the mass range around the isotope clusters with a net charge of -9. (B) Deconvoluted spectrum obtained using MaxEnt.

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As can be seen from Table 1, the maximum errors obtained on the mass determination of these long oligonucleotides are (50 ppm; in the molecular mass range of these products (35 00037 000 Da), this corresponds to an error of (1.8 Da. It was calculated that, taking the 100 T tail into account and restricting the length of the possible oligonucleotides to the expected length, only four different base compositions were possible within the mass range of the error around the observed masses with a number of bases equal to the expected number of bases (one of them being the expected base composition). Without restriction to the number of bases, seven possible base compositions were calculated using the spreadsheet. Analysis of the Short Oligonucleotides (18-27-mers) Using CZE-ES-Q-TOFMS. These short, biotinylated oligonucleotides were analyzed using the same conditions. As can be seen from the analysis of R8958 (Figure 4), CZE is extremely efficient in removing all metal ion adducts from the oligonucleotide. Without taking any supplementary precautions to avoid these salt adducts, no metal ion adducts at all were detected. In this spectrum, the following products could be identified: MM ) 5518.85 Da, the debiotinylated oligonucleotide; MM ) 5595.85 Da, the biotinylated oligonucleotide missing one G residue; MM ) 5632.85 Da, the biotinylated oligonucleotide missing one C residue; MM ) 5790.85 Da, the biotinylated oligonucleotide which lost an adenine base, probably due to depurination; and MM ) 5922.85 Da, corresponding to the expected R8958. As can be seen from the analysis of R8633 (Figure 5), CZE-ES-Q-TOFMS can also be used for the identification of oligonucleotides with the same length, only differing by one base in their sequence. It is obvious from these data that even the smallest difference (A to T switch, differing 9 Da in mass) can be detected without any problem. This makes the quality control of wobbles possible. These wobbles, having the same length, cannot be analyzed by HPLC or gel electrophoretic techniques, since these techniques are based on the separation by length differences. As can be seen from Table 1, the maximum errors obtained on the mass determination of the short oligonucleotides are (100 ppm; in the molecular mass range of these oligonucleotides (6000-9000 Da), this corresponds to an error of (0.8 Da. Calculation of the possible base compositions with a molecular mass within the mass range of the error around the observed masses, and with a number of bases equal to the expected number of bases, showed only one possibility, being the expected base composition. Without the restriction to the number of bases, two possible base compositions were calculated using the spreadsheet.

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The molecular masses observed for R7081, R6963, and R6841 (from Pharmacia) were 87.9 Da higher than their expected masses and than the observed masses for identical oligonucleotides obtained from SGS. This is probably due to a different chemistry used in the synthesis of these biotinylated oligonucleotides. CONCLUSION These experiments prove CZE to be a very effective interface for the analysis of oligonucleotides on electrospray mass analyzers. The use of electrophoresis is very efficient in removing all metal ion adducts without any preliminary sample treatment, thus greatly enhancing the sensitivity and the precision of the molecular mass determination.6 The use of the CZE-ES-Q-TOF system not only enhances the sensitivity by a factor of 20 compared to that of conventional quadrupole electrospray, but it also has a superior mass resolution.18 As a result, the deconvoluted data exhibit high precision ((50 ppm for the long oligonucleotides) and high resolution (peak widths at half-height of 4 Da). An explanation for the higher relative precision obtained for the long oligonucleotides ((50 ppm) compared to the short oligonucleotides ((100 ppm) may be found in the observation that the raw spectra of the long oligonucleotides have 4 times more peaks than the short oligonucleotides. Thus, having more measurement points, the nonsystematic errors in the mass assignment of each peak in the raw spectrum will compensate each other better in the deconvoluted spectrum of the long oligonucleotides. Note, however, that the absolute precision obtained for the short oligonucleotides ((0.8 Da) is, as would be expected, better than that for the long oligonucleotides ((1.8 Da). These characteristics make CZE-ES-Q-TOFMS a very useful tool in the quality control of oligonucleotides, e.g., for the identification of oligonucleotides, for the detection of failed sequences and other impurities or degradation products (debiotinylated primers), and for the determination of the base composition. Further experiments are being performed to investigate the use of this system for the characterization of PCR products. The CZE separation step will be optimized to separate the enzymes, dNTPs, salts, and primers from the PCR product.

Received for review February 10, 1998. Accepted May 7, 1998. AC980147X