Anal. Chem. 2002, 74, 226-231
Improvement in the Apparent Mass Resolution of Oligonucleotides by Using 12C/14N-Enriched Samples Kai Tang,* Mona Shahgholi,† Benjamin A. Garcia, Paul J. Heaney, and Charles R. Cantor
Sequenom Inc., 3595 John Hopkins Court, San Diego, California 92121 Lincoln G. Scott and James R. Williamson
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
The apparent mass resolution of oligonucleotides in timeof-flight (TOF) mass spectrometers has been examined. In a reflectron TOF instrument, where the isotopic profile can be completely resolved, the apparent resolution matches the instrument’s resolving power. In a linear TOF instrument, unresolved isotopic profiles limit the apparent resolution to much lower values than the actual instrument resolution. By using 12C/14N-enriched oligonucleotides, the apparent resolution can be improved significantly. The isotope enrichment method also enhances the signal-to-noise ratio. With the introduction of ionization methods for analysis of large molecules, the need for accurate mass assignments has prompted studies on isotopic distributions.1-5 Theoretical calculations and computer algorithms have been developed for the simulation of isotope profiles.1,2,6-11 At the same time, efforts to improve the resolving power of mass spectrometers have resulted in significant advances in instrumentation. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) coupled with various mass analyzers have made it possible to resolve large molecules at unit mass resolution. Isotopic resolution for large * To whom correspondence should be addressed. Phone: 858-202-9089. Fax: 858-202-9001. E-mail:
[email protected]. † Current address: Department of Chemistry, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125. (1) Yergey, J.; Heller, D.; Hansen, G.; Cotter, R. J.; Fenselau, C. Anal. Chem. 1983, 55, 353-356. (2) Yergey, J. A. Int. J. Mass Spectrom. Ion Phys. 1983, 52, 337-349. (3) Beavis, R. C. Anal. Chem. 1993, 65, 496-497. (4) McLafferty, F. W. Acc. Chem. Res. 1994, 27, 379-386. (5) O’Connor, P. B.; Little, D. P.; McLafferty, F. W. Anal. Chem. 1996, 68, 542-545. (6) Zubarev, R. A. Int. J. Mass Spectrom. Ion Processes 1991, 107, 17-27. (7) Zubarev, R. A.; Demirev, P. A.; Håkansson, P.; Sundqvist, B. U. R. Anal. Chem. 1995, 67, 3793-3798. (8) Senko, M. W.; Beu, S. C.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1995, 6, 229-233. (9) Rockwood, A. L.; Van Orden, S. L.; Smith, R. D. Anal. Chem. 1995, 67, 2699-2704. (10) Grange, A. H.; Brumley, W. C. J. Am. Soc. Mass Spectrom. 1997, 8, 170182. (11) Zubarev, R. A.; Demirev, P. A. J. Am. Soc. Mass Spectrom. 1998, 9, 149156.
226 Analytical Chemistry, Vol. 74, No. 1, January 1, 2002
protein ions can be obtained not only on magnetic sector and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers, but also on reflectron time-of-flight (TOF) mass spectrometers with delayed extraction. However, in MALDI, it is more difficult to resolve nucleic acids to the same degree as for proteins of similar mass, presumably because of lower ionization efficiency and higher probability of ion fragmentation. Until now, high-resolution MALDI spectra for nucleic acids of 25 nucleotides have been reported only by using FTMS.12 In TOF mass spectrometers, most heavy DNA ions undergo metastable decay in the flight tube, making it difficult to analyze them in the reflector mode. In the linear mode, the instrument resolution is not high enough to resolve the isotopic profiles. Herein, we define the instrument resolution as the intrinsic resolving power of a mass spectrometer. In TOF instruments, it is determined by physical parameters, such as the length of the flight tube, target and extraction voltages, extraction delay time, and ion source dimensions. The instrument resolution is independent of the nature of the analyte molecules. The apparent resolution is the measured resolution of m/∆m, where ∆m is the full width at half-maximum (fwhm). Various factors can affect the apparent resolution, including adduct formation, metastable decay of ions, space charge in the ion cloud, and initial energy and spatial distribution of ions. These factors may be controlled experimentally by desalting the sample, use of a cool matrix, or time-lag focusing. One important factor that directly influences the apparent resolution and cannot be readily controlled is the isotopic distribution of the analyte molecules. In the absence of other factors affecting the apparent resolution, if the instrument resolution is not sufficiently high to resolve individual isotopic peaks at half-maximum, the apparent resolution will be significantly lower, since ∆m will include contributions from several isotopic peaks. For oligonucleotides, the main source of high mass isotopes is 13C, which accounts for 1.1% of all natural carbon. Therefore, a 10-nucleotide DNA with about 100 carbon atoms should have one 13C on average. Therefore, the most probable isotope peak is no longer the monoisotopic peak, but rather the peak is 1 Da higher. (12) Li, Y.; Tang, K.; Little, D. P.; Ko ¨ster, H.; Hunter, R. L.; McIver, R. T. Anal. Chem. 1996, 68, 2090-2096. 10.1021/ac010804c CCC: $22.00
© 2002 American Chemical Society Published on Web 12/01/2001
As the length of the oligonucleotide increases, the most probable isotope peak shifts to higher masses, and the spread of the isotopic profile increases. By using 12C-enriched oligonucleotides, the 13C contribution to the isotope profile is reduced, thereby resulting in an increased intensity of the monoisotopic peak and a narrower isotopic distribution. This can significantly improve the apparent mass resolution when the instrument resolution is not high enough to resolve the isotope profile. Double-depletion of 13C and 15N for protein samples has been performed to reveal the monoisotopic peak for accurate mass assignment in ESI-FTMS.13 The ultrahigh resolution of FTMS made it easy to fully resolve each isotopic peak. In this report, we examine the implication of such heavy-isotope depletion in oligonucleotide analyses in TOF mass spectrometers where resolution is not always high enough to fully resolve the isotope profile. EXPERIMENTAL SECTION Natural deoxynucleotides 16-nt (5′-TCC CTT ATT TCC CTC A), 17-nt (5′-CCA TCC ACT ACA ACT AC), and 28-nt (5′-CCA TCC ACT ACA ACT ACA TGT GTA ACA G) were purchased from Operon Technologies (Alameda, CA), and were used without purification. 3-Hydroxypicolinic acid (3-HPA) (MALDI grade) was obtained from Fluka (Milwaukee, WI). Synthesis of Nucleoside Triphosphates. Isotopically depleted nucleoside triphosphates were prepared from cellular extracts of Methylophilus methylotrophus by standard methods, with the exception that (12C, 99.95%) methanol (Cambridge Isotopes Laboratories, Andover, MA) was included in the growth media.14 Briefly, M. methylotrophus (requires methanol as carbon source) is grown on minimal medium containing 12C-enriched methanol. The cells are harvested and lysed by detergent, the proteins are removed by phenol/chloroform extraction, and the total nucleic acid fraction is purified using 2-propanol precipitation. The nucleic acids are digested to mononucleotides using nuclease P1, and the deoxy and ribonucleotides are separated using a boronate affinity column. The ribonucleoside monophosphates are then enzymatically converted to triphosphates that are used for in vitro transcription of the desired RNA sequence. The cost of making the 12C-enriched nucleotides is comparable to the 12C-depleted nucleotides commercially available. The quality of the enriched nucleotides can be verified by electrospray ionization (ESI) mass spectrometry. In Vitro Transcription of RNA. 12C-enriched RNA was synthesized by in vitro transcription with T7 RNA polymerase from an oligonucleotide template using a single-stranded template region and a double-stranded promoter region.15 Transcription reactions were carried out under the following conditions: 80 mM K-Hepes (pH 8.1), 1 mM spermidine, 10 mM dithiothreitol, 0.01% Triton X-100, 80 mg/mL polyethyene glycol, 10 mM MgCl2, 4 mM each nucleoside triphosphate, 1 µM template DNA, 1 µM promoter DNA, 2 unit/mL inorganic pyrophosphatase (Sigma, St. Louis, (13) Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan, T. M. J. Am. Chem. Soc. 1997, 119, 433-434. (14) Batey, R. T.; Battiste, J. L.; Williamson, J. R. Methods Enzymol. 1995, 261, 300-322. (15) Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C. Nucleic Acids Res. 1987, 15, 8783-8798.
MO), 80 units/mL RNAse inhibitor (Promega Corp., Madison, WI), and 3000 units/mL T7 RNA polymerase. The reaction was incubated for 4 h in a water bath at 37 °C, quenched with 0.1 volume of 0.5 M EDTA (pH 8.0), and extracted with an equal volume of phenol/chloroform (Fisher Scientific, Tustin, CA) equilibrated with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The aqueous layer was ethanol-precipitated with the addition of 0.33 volume of 10 M ammonium acetate and 3.5 volumes of 100% ethanol for 2 h at -20 °C. The RNA precipitate was desalted, and unincorporated nucleoside triphosphates were removed using 10 mL of double-distilled H2O in a 3500 MWCO Centricon (Millipore Corp., Bedford, MA). Mass Spectrometry. Standard matrix solutions were prepared by dissolving 35 mg of 3-HPA and 7.14 mg of diammonium citrate in 1 mL of 10% acetonitrile and water. Matrix solutions were filtered with syringe filters (0.2-µm i.d.) from Alltech Inc. (Deerfield, IL) prior to dispensing to remove any insoluble particles. About 0.15 µL of the matrix solution was pipetted onto a stainless steel target and allowed to dry. Aqueous analyte solution from 1 to 10 µM of the same volume was then pipetted on top of the matrix spot and dried under ambient conditions. Mass spectra were obtained on a Voyager DE-PRO TOF mass spectrometer from Applied Biosystems (Framingham, MA). Instrument parameters were set as follows: in linear mode, accelerating voltage +20 kV, grid voltage +18.9 kV, delay 400450 ns, guide wire 0.08% of the accelerating voltage; in reflectron mode, accelerating voltage +20 kV, grid voltage +14.84 kV, delay 200-300 ns, and guide wire 0.002% of the acceleration voltage. The reflector voltage was set as 1.12× the acceleration voltage. The Voyager DE-PRO was equipped with a digitizing oscilloscope (TDS 520D, Tektronics). The sampling bin size was set at 2 ns in the linear mode and 1 ns in the reflector mode, with an input bandwidth of 500 MHz. The vertical sensitivity was set at 200 mV full-scale in linear mode and 50 mV in reflector mode. All of the spectra presented here were raw spectra obtained using 10-20 laser shots in the linear mode and at least 50 laser shots in the reflector mode. Computer simulations for all of the spectra were performed using IsoPro 3.0.16 For most simulations, 5′-OH DNAs of desired lengths were used. RESULTS In current state-of-the-art reflectron TOF mass spectrometers, the instrument resolution is higher than 10 000 (fwhm), which is usually high enough to fully resolve the isotope profile in the low mass (
