Anal. Chem. 1996, 68, 2090-2096
High-Resolution MALDI Fourier Transform Mass Spectrometry of Oligonucleotides Yunzhi Li,† Kai Tang,‡ Daniel P. Little,‡ Hubert Ko 1 ster,§ Richard L. Hunter,† and Robert T. McIver, Jr.*,⊥
IonSpec Corporation, 18009-F Skypark Circle, Irvine, California 92714, Sequenom Inc., 101 Arch Street, Boston, Massachusetts 02110, Department of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany, and Department of Chemistry, University of California, Irvine, California 92717
The matrix-assisted laser desorption/ionization (MALDI) method has been used with an external ion source Fourier transform mass spectrometer (FTMS) to analyze singlestranded, mixed-base oligomers of DNA. It is demonstrated that ultrahigh mass resolution (830 000 fwhm) can be achieved for small oligomers, and high resolution (136 000 fwhm) can be achieved for a 25-mer at m/z 7634. MALDI-FTMS can clearly separate the molecular ion peaks from analyte-matrix adduct peaks and alkali metal-containing species that result from replacement of hydrogen ions with sodium or potassium ions at multiple sites along the phosphate backbone. Previous MALDIFTMS studies of oligonucleotides had two limitations: (1) low sensitivity due to difficulty in trapping the high kinetic energy ions made by the laser and (2) fragmentation of the ions due to the long delay (tens to hundreds of milliseconds) between their formation and detection. Both of these problems are alleviated in the present study. With the external ion source FTMS instrument, ions made by MALDI are injected at low energy into the analyzer cell by a rf-only quadrupole ion guide, captured by gating the voltage on the trapping plates, and cooled by a 0.5-s pulse of argon gas. Under these conditions, fragmentation is minimized, and DNA ions can be trapped in the FTMS analyzer cell for greater than 50 s. Sensitivity is also improved, as demonstrated by detection of 1 pmol of a single-stranded, mixed-base 20-mer of DNA, with a signalto-noise ratio greater than 20:1. Since its introduction by Karas and Hillenkamp in 1988,1 matrix-assisted laser desorption/ionization (MALDI) has become a powerful technique to produce gas-phase ions of large molecules to be subsequently analyzed by mass spectrometry. Usually, MALDI is coupled with a time-of-flight (TOF) mass spectrometer due to the unlimited mass detection range of these instruments.2 However, since the ions produced by MALDI have broad energy distributions,3,4 significant peak broadening occurs, which results in low mass resolution. Even with reflectron TOF instruments, †
IonSpec Corp. Sequenom Inc. § University of Hamburg. ⊥ University of California, Irvine. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (2) Cotter, R. J. Anal. Chem. 1992, 64, 1027A-1039A. (3) Beavis, R. C.; Chait, B. T. Chem. Phys. Lett. 1991, 181, 479-484. (4) Pan, Y.; Cotter, R. J. Org. Mass Spectrom. 1992, 27, 3-8. ‡
2090 Analytical Chemistry, Vol. 68, No. 13, July 1, 1996
which use a retarding electric field to turn the ions around in the flight path to achieve energy focusing, peak broadening still persists. Recent TOF experiments employing a time lag focusing method, initially described in 1955 by Wiley and McLaren,5 have achieved better energy focusing6-8 and thus better resolution over a narrow range of the mass spectrum (e.g., 12 500 for bovine insulin9 ). Another factor that limits mass resolution is metastable decay caused by excessive energy transferred to the analyte ions. In this regard, DNA is one of the most fragile biomolecules that can be analyzed by MALDI-MS. Except for thymidine, the DNA bases are easily protonated in an acidic environment such as the MALDI matrix, and this likely catalyzes the cleavage of the N-glycosidic bond.10,11 Due to their higher proton affinity, the purine bases, especially guanosine, are very susceptible to hydrolysis. As a result, depurination is usually observed in MALDI-TOF spectra when the mass resolution is high enough to make such an identification. Moreover, the energy transferred to analyte molecules during MALDI is usually high enough to cause fragmentation promptly after their desorption. When fragmentation occurs in the acceleration region of the TOF instrument, the mass resolution is seriously degraded because the acceleration potential of the fragment ions depends on the position where the metastable decay occurs.12 The need for improved mass resolution has encouraged efforts to couple MALDI with other types of mass spectrometers. In 1991, Hettich and Buchanan showed that Fourier transform mass spectrometry (FTMS) was useful for analysis of normal and modified oligonucleotides from dimers to hexamers.13,14 Abundant (M - H)- ions were observed, but there was also extensive (5) Wiley, W. C.; McLaren, I. H. Rev. Sci. Instrum. 1955, 26, 1150-1157. (6) Colby, S. M.; King, T. B.; Reilly, J. P. Rapid Commun. Mass Spectrom. 1994, 8, 865-868. (7) Brown, R. S.; Lennon, J. J. Anal. Chem. 1995, 67, 1998-2003. (8) Whittal, R. M.; Li, L. Anal. Chem. 1995, 67, 1950-1954. (9) Vestal, M. L.; Juhasz, P.; Martin, S. A. Rapid Commun. Mass Spectrom. 1995, 9, 1044-1050. (10) Nordhoff, E.; Cramer, R.; Karas, M.; Hillenkamp, F.; Kirpikan, F.; Kristiasen, K.; Roepstorff, P. Nucleic Acids Res. 1993, 21, 3347-3357. (11) Tang, K.; Allman, S. L.; Chen, C. H. Rapid Commun. Mass Spectrom. 1993, 7, 943-948. (12) Nordhoff, E.; Karas, M.; Cramer, R.; Hahner, S.; Hillenkamp, F.; Kirpekar, F.; Lezius, A.; Muth, J.; Meier, C.; Engels, J. W. J. Mass Spectrom. 1995, 30, 99-112. (13) Hettich, R. L.; Buchanan, M. V. J. Am. Soc. Mass Spectrom. 1991, 2, 402412. (14) Hettich, R. L.; Buchanan, M. V. Int. J. Mass Spectrom. Ion Processes 1991, 111, 365-380. S0003-2700(96)00126-6 CCC: $12.00
© 1996 American Chemical Society
fragmentation. Although subsequent experiments showed that the extent of fragmentation was influenced strongly by the matrix, generally FTMS gave significantly more fragmentation than MALDI-TOF.15 Hettich and Buchanan explained this by suggesting that the ions undergo extensive metastable decay during the relatively long times (tens to hundreds of milliseconds) required for FTMS detection. Their recent results have extended the upper mass range to 12-mers and achieved a mass resolution of 1700 for a hexamer.16 Higher mass resolution for MALDI coupled with FTMS was demonstrated in 1992 by Wilkins and co-workers.17 They achieved resolutions greater than 50 000 for some low molecular weight peptides and 11 580 for bovine insulin. However, this level of performance has never been demonstrated for MALDI of oligonucleotides. To date, therefore, results have suggested that DNA ions produced by MALDI have short lifetimes which prevent them from being analyzed by FTMS. A different type of FTMS apparatus, employing an external MALDI ion source, was developed in 1994 by McIver and coworkers.18-23 With this method, ions are generated in a MALDI source that is outside the magnetic field, about 1 m from the FTMS analyzer cell. The ions are extracted from the source and focused by a radio frequency (rf)-only quadrupole ion guide that transports them to the analyzer cell.24,25 The external ion source FTMS instrument has achieved the highest mass resolution ever demonstrated for biomolecules made by MALDI. Human insulin at m/z 5808 has been detected at a mass resolution of 850 000, and the protein cytochrome c at m/z 12 360 has been detected at a resolution of 81 000.26 The purpose of this investigation was to explore the possibility of applying the external ion source FTMS method to highresolution analysis of oligodeoxynucleotides. Of particular concern was the role that metastable decay would play in limiting the mass resolution. With TOF instruments, ions made by MALDI are detected less than a millisecond after the laser pulse, but with the external ion source FTMS instrument, the delay time is typically greater than 5 s. This is even longer than the “insource ionization” FTMS experiments done by Hettich and Buchanan. Another concern was detection sensitivity and whether ions made by MALDI with a wide range of kinetic energies could be efficiently transported by the rf-only quadrupole ion guide and trapped in the FTMS analyzer cell. In this report, we demonstrate that greatly improved sensitivity and mass resolution for DNA oligomers can be achieved with the external ion source FTMS method. Even though the time scale (15) Stemmler, E. A.; Hettich, R. L.; Hurst, G. B.; Buchanan, M. V. Rapid Commun. Mass Spectrom. 1993, 7, 828-836. (16) Stemmler, E. A.; Buchanan, M. V.; Hurst, G. B.; Hettich, R. L. Anal. Chem. 1995, 67, 2924-2930. (17) Castoro, J. A.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. J. Am. Chem. Soc. 1992, 114, 7571-7572. (18) McIver, R. T., Jr.; Li, Y.; Hunter, R. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4801-4805. (19) Li, Y.; Hunter, R. L.; McIver, R. T., Jr. Nature 1994, 370, 393-395. (20) McIver, R. T., Jr.; Li, Y.; Hunter, R. L. Int. J. Mass Spectrom. Ion Processes 1994, 132, L1-L7. (21) McIver, R. T., Jr.; Li, Y.; Hunter, R. L. Rapid Commun. Mass Spectrom. 1994, 8, 237. (22) Li, Y.; McIver, R. T., Jr.; Hunter, R. L. Anal. Chem. 1994, 66, 2077-2083. (23) Li, Y.; McIver, R. T., Jr. Rapid Commun. Mass Spectrom. 1994, 8, 743749. (24) McIver, R. T., Jr. U.S. Patent 4,535,235, 1985. (25) McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Processes 1990, 98, 35-50. (26) Li, Y.; Hunter, R. L.; McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Processes, in press.
for detection of the ions is very long, extensive fragmentation is not observed because the ions are cooled by a pulse of argon gas. In all the cases investigated, stable (M + H)+ positive ions or (M - H)- negative ions are the base peaks in the mass spectra. In addition, it is shown that high mass resolution (>100 000) and high mass measurement accuracy (
