Molecular Weight Determination of Underivatized Oligodeoxyribonucleotides by Positive-ion Matrix-assisted Ultraviolet Laser-desorption Mass Spectrometry Bernhard Spengler, Ying Pan and Robert J. Cotter' Department of Pharmacology and Molecular Sciences, Middle Atlantic Mass Spectrometry Facility, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Lou-Sing Kan Department of Biochemistry, Division of Biophysics, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA
A Wiley-McLaren type time-of-flight mass spectrometer has been used for molecular weight measurements of several unprotected oligodeoxyribonucleotides using matrix-assisted UV laser desorption. Approximately 10 to 100 pmol of sample was required for recording their positive-ion mass spectra with a mass resolution in the range of 150 to 300 (Full width at half maximum) (FWHM). Little fragmentation was observed.
Ultraviolet laser-desorption mass spectrometry (UVLD-MS) has become increasingly important as a method for the sensitive analysis of large bio-organic substances following the development of the UV-absorbing matrix-assisted technique. This technique, originally used for determining the molecular weights of has been reported to be applicable to the analysis of polynucleotides as well." Recently, we developed a UVLD-MS instrument' which employs a Wiley-McLarenll type time-of-flight mass analyzer. That instrument was used for molecular weight measurements of proteins up to more than 100 kDa; and in this paper we have utilized the same instrument for the analysis of several oligodeoxyribonucleotides. This technique shows some interesting differences between the matrix-assisted UVLD-MS of oligonucleotides and proteins, and is different as well from oligonucleotide analysis with other desorption techniques. While the mass spectrometric analysis of oligonucleotides using a variety of soft ionization techniques has been reported, they present an interesting analytical challenge because of the presence of multiple negative charges on the connecting phosphate groups. Thus, they are most easily analyzed from the negative-ion mass spectra of derivatized or (in the case of synthetic oligonucleotides) as fully protected samples. McNeal et a/.'*,l3 employed negative-ion plasma desorption mass spectrometry (PD-MS) for the analysis of fully protected oligonucleotides up to the heptamer, while more recently, oligonucleotides with up to 9 protected units have been detected by PD-MS in the positive-ion mode.I4 In the latter study, the amount of analyte consumed after recovery of the analyzed sample was about 1.4 nmol. Negative-ion fast-atom bombardment (FAB) mass spectral analyses of both protectkd and unprotected oligonucleotides have been demonstrated . ' ~ recently with sensitivities of about 10 n m ~ l . ' ~Most ion spray analysis of oligonucleotides in the negativeion mode with a sensitivity of 200pmol has been reported." 'Author to whom correspondence should be addressed.
AI mirror
Quantel
YG 660 quartz lens &$ .. I
I
I sample probe
,drift tube
1
n
detector
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I PC-AT I
data system
Figure 1. Schematic diagram of the UV-laser desorption/ionization mass spectrometer.
The positive-ion matrix assisted UV laser-desorption mass spectra of peptides and proteins are characterized by singly-charged (protonated) molecular ions, in contrast to the multiply-charged ions of electrospray spectra and the cationized (i.e., MNa') species frequently observed in FAB- and PD-MS spectra. In addition to the efficient interaction of the UV laser irradiation with the highly abundant matrix molecules (generally, nicotinic acid), the resulting electronically excited matrix molecules and ions may also serve as a rich source of protons, which may be transferred to relatively acidic molecules. Thus, our approach was to examine the positive-ion matrix-assisted UV laser-desorption mass spectra of synthetic oligonucleotides. In general, the matrix technique works best when the analyte shows vanishing spectral absorption at the wavelength used for excitation of the matrix. However, as can be seen from the data presented here, nucleotides produce good results as well, despite their relatively high absorptivity at 266 nm. EXPERIMENTAL Mass spectra were obtained on a modified CVC Products (Rochester, NY, USA) model 2000 time-offlight mass spectrometer, described in detail This instrument is shown schematically in Fig. 1. A Quantel (Santa Clara, CA, USA) model YG-660
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frequency-quadrupoled Nd:YAG laser (266 nm, 5 ns pulse' duration) has been used for ion desorption. The laser beam has been focused onto the sample surface by a quartz lens of 300 mm focal length to a spot diameter of about 150 pm. In contrast to other laser-desorption mass spectrometer^',^ used for matrix-assisted UVLD-MS, desorbed ions were not accelerated promptly, but were extracted after a suitable delay of several microseconds to allow time focusing. Ions are post-accelerated into a venetian-blind type conversion dynode, with secondary electrons detected by a Thorn/EMI (Fairfield, NJ, USA) electron multiplier. Mass spectra were recorded and stored in a LeCroy (Spring Valley, NY, USA) model 9400A digital oscilloscope/transient recorder and signal-averaged by PC-based software. All the oligodeoxynucleotides used in this report were synthesized using phosphite-triester chemistry'' for previous nuclear magnetic resonance (NMR) studies.2"-22The sodium counter-ion was replaced with ammonium using a D E A E column. Oligonucleotides were prepared for mass spectrometric analysis by dissolving the samples in ultra-pure water. Useful concentrations were found in the range of to M. An aqueous nicotinic acid solution of 5x molar concentration was used. 1 pL of analyte solution and 1pL of matrix solution were mixed on a silver probe tip and blow dried. RESULTS AND DISCUSSION Eight oligodeoxyribonucleotides, containing between 3 and 8 nucleotide units were investigated by matrixassisted UV laser-desorptionlionization mass spectrometry. Their general structure is shown in Fig. 2 and does not include a phosphate group at the 5' end. The oligonucleotides studied included A-A-G (mol.wt [free acid] 893), T-G-G (mol.wt 900), C-C-A-A (mol.wt 1142), C-G-C-G (mol.wt 1174), T-T-G-G (mol.wt 1204), C-C-A-A-T (mol.wt 1446), C-T-T-G-G (mol.wt
0 0
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Figure 2. General structure of synthetic oligodeoxyribonucleotides used in this study.
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Figure 3. Matrix-assisted UV-laser desorption mass spectrum of 40 pmol of the oligodeoxyribonucleotide T-T-G-G (mol.wt 1204). The molar ratio of analyte to matrix was 1:1250. [Calculated MH' = 1204.2 (monoisotopic mass) 1204.8 (average mass)]. The molecular ion intensity is approximately 8% of the base peak at m / z 203 originating from the nicotinic acid matrix.
1493) and T-C-T-T-G-G (mol.wt 1797). All samples examined showed intense signals for the protonated parent molecule. For comparison, non-matrix-assisted UV laser-desorption spectra were obtained for several oligodeoxyribonucleotides, by drying the analyte solution onto a silver substrate without addition of matrix. This method, known to give good results for various types of compounds in the mass range below 2000 Da,23 did not provide significant ion signals in the case of the oligonucleotides. Mass calibration was carrried out using known mass peaks from the nicotinic acid matrix. The observed molecular weights of the oligonucleotides in all cases were found to be within +1 mass unit of their calculated monoisotopic molecular weights. Figure 3 shows the matrix-assisted UV laserdesorption mass spectrum of the oligodeoxyribonucleotide T-G-G-G. 1.OpL of a 4 x 1 0 - ' ~solution mixed with 1 pL of the nicotinic acid solution was used, corresponding to 40pmol of sample and a molar ratio of sample to matrix of 1:1250. The mass spectrum reveals several differences from those of proteins obtained on the same instrument with the same preparation technique.' First of all, matrix-specific ions of high intensity are observed. In addition, the quasimolecularion region shows strong asymmetric peak broadening above the mass of the protonated molecule. While such broadening might be attributed in part to the presence of unresolved adduct ions, it is noteworthy that lowering the potential of the post-accelerating dynode reduces the effect. As noted before, nucleotides have a strong absorption at 266 nm, whereas peptides show only minor spectral absorption at this wavelength. Peak broadening may thus be due to decay of internally excited metastable ions after acceleration and prior to post-accceleration. The electron multiplier would then detect neutral fragments of decayed parent molecules at a later time than post-accelerated quasimolecular ions. Using a wavelength off the absorption band of nucleotides, such as the frequency tripled Nd:YAG
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wavelength at 355 nm, in combination with a suitable matrix for this wavelength should lead to a lower internal excitation of the nucleotide molecules and thus to a lower degree of metastable-ion decay. Except for the losses of a guanine base for T-G-G and T-T-G-G, fragment ions were not observed in the matrix-assisted UV-laser desorption mass spectra of oligonucleotides. All other signals observed below the quasimolecular-ion regions are due to known photochemical oligomers and fragments of the nicotinic acid matrix. The mass resolution in matrix-assisted UV-laser desorption of high-mass molecules is known to be limited by the formation of unresolved adduct ions and other specific processes, rather than the inherent resolution of the i n ~ t r u m e n tIn . ~ contrast to recent analyses of large proteins with masses up to 118 000 Da, done on the identical instrument,' the mass resolving power for oligodeoxyribonucleotides of masses below 2000 Da was as high as observed for infra red (IR) laser-desorption18 and non-matrix-assisted UV laserdesorption, i.e., in the range of 150 to 300 measured as full width at half maximum (FWHM). The instrumental mass resolving power is high enough to resolve protonated, cationized and matrix-attached molecules in this mass range. In contrast to the analysis of proteins, intense signals of matrix-attached analyte ions were not observed. In addition to the protonated parent molecule, the most prominent quasimolecular ions observed (in a few cases) were those correspnding to attachment of ammonium or alkali ions. Figure 4 shows the molecular-ion region of the oligodeoxyribonucleotide C-C-A-A (mol.wt 1142), in which the protonated molecular ion is observed with a mass resolving power of about 285, (FWHM). Figure 5 shows the mass spectrum of C-G-C-G (mol.wt 1174), for which a strong peak corresponding to the MNH: ion is also observed. Since attachment of the parent molecule by matrix molecules or cations affects the mass resolution (and mass accuracy) when high-mass peaks are not resolved, the lack of attachment of nicotinic acid to 100
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Figure 5. Matrix-assisted UV-laser desorption mass spectrum of 100 pmol of the oligodeoxyribonucleotide C-G-C-G (mol.wt 1174). The molar ratio of analyte to matrix was 1500. [Calculated MH' = 1174.2 (monoisotopic mass), 1174.2 (average mass)].
oligonucleotides suggests, that analysis of larger polynucleotides might lead to higher effective mass resolution, compared to protein analysis, where attachment by nicotinic acid is ~ o m m o n . ~ , ~ In contrast to the other samples tested, C-G-C-G is a self-complementary oligonucleotide, capable of forming double-stranded molecules with itself (duplexes) at room temperature in 0.1 M neutral aqueous solution. However, the intensity of the [2M+H]+ ion was not higher than for the other oligonucleotides, indicating that duplexes of C-G-C-G have either not been desorbed intact or their formation has been disturbed by the highly abundant nicotinic acid. The formation of oligomeric ions of the parent molecules is, however, strongly affected by the analyte concentration in the analyte/matrix mixture, as observed for peptides as well. Figure 6 shows a spectrum of T-T-G-G for which 100
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Figure 4. Matrix-assisted UV-laser desorption mass spectrum of 100 pmol of the oligodeoxyribonucleotide C-C-A-A (mol.wt 1142). The molar ratio of analyte to matrix was 1:500. [Calculated MH+ = 1142.2 (monoisotpic mass), 1142 (average mass)].
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the concentration of the analyte in this case was 1.4 x ' ~ O - ~molar corresponding to 200 pmol of analyte and a molar ratio of analyte to matrix of only 1:350. The intensity of dimeric (duplex) ions is much higher compared to the value in the spectrum in Fig. 3, and the peaks ai-e much broader. Samples with analyte molar resulted in concentrations of more than only weak signals of the analyte, comparable to those in the spectra taken with non-matrix-assisted .UV laser desorption. Conclusions Positive-ion matrix-assisted UV laser-desorption provides an easy, fast and sensitive method of moleular weight determination of synthetic, unprotected oligonucleotides. Derivatization was not necessary to compensate for the large number of negative charges. Positively charged ions were detected easily, despite the highly anionic character of the analyte and in contrast to analysis by other mass spectrometric iondesorption techniques. Acknowledgements This work was supported by grants DIR 86-10589 from the National Science Foundation and GM 34252 (to LSK) from the National Institutes of Health. B. Spengler was supported by a Deutsche Forschungsgemeinschaft Fellowship. We also thank A. Ono for technical assistance. Mass spectral analyses were carried out at the Middle Atlantic Mass Spectrometry Laboratory, an NSF supported Shared Instrumentation Facility. REFERENCES 1. M. Karas and F. Hillenkamp, Anal. Chem. 60, 2299 (1988). 2. M. Karas, U. Bahr, A. Ingendoh and F. Hillenkamp Angew. Ckem. l n f . Ed. En@" 28, 760 (1989). 3. M. Karas, U. Bahr and F. Hillenkamp, Int. J. Mass Spectrom. Ion Proc. 92, 231 (1989).
4. M. Karas, A. Ingendoh, U. Bahr and F. Hillenkamp, Biomed. Enuiron. Mass Spectrom. 18, 841 (1989). 5. R. C. Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3, 233 (1989). 6. R. C. Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3 , 432 (1989). 7. R. C . Beavis and B. T. Chait, Rapid Commun. Mass Spectrom. 3 , 436 (1989). 8. M. Salehpour, I. Perera, J. Kjellberg, A. Hedin, M. A. Islamian, P. Hakansson and B.U.R. Sundqvist Rapid Commun. Mass Spectrom. 3 , 259 (1989). 9. B. Spengler and R. J. Cotter, Anal. Chem. (in press). 10. F. Hillenkamp, M. Karas, A. Ingendoh and B. Stahl, Proceedings of the Second International Symposium on Mass Spectrometry in the Health and Life Sciences, San Francisco, ed. by, A. Burlingame and J. A. McCloskey, Elsevier (in press). 11. W. C. Wiley and I. H. McLaren, Rev. Sci. Instrumen. 26, 1150 (1955). 12. C. J. McNeal, K. K. Ogilvie, N.Y. Theriault and M. J. Nemer, J . Am. Chem. SOC. 104, 981 (1982). 13. C. J. McNeal and R . D. Macfarlane, J . A m . Chem. SOC. 103, 1609 (1981). 14. A. Viari, J . P. Ballini, P. Meleard, P. Vigny, P. Dousset, C. Blonski and D. Dhire, Biomed. Enuiron. Mass Spectrom. 16, 225 (1988). 15. D. Griffin, J. Laramee, M. Deinzer, E . Stirchak and D. Weller Biomed. Enuiron. Mass Spectrom. 17, 105 (1988). 16. L. Grotjahn, H. Bloecker and R. Frank, Biomed. Mass Spectrom. 12, 514 (1985). 17. T. R. Covey, R. F. Bonner, B. I. Shushan and J. Henion, Rapid Commun. Mass Spectrom. 2, 249 (1988). 18. J . K. Olthoff, I. Lys, P. Demirev and R. J. Cotter, Anal. Instrumen. 16, 93 (1987). 19. P. S. Miller, D. M. Cheng, N. Dreon, K. Jayaraman, L.-S. Kan, E . E. Leutzinger, S. M. Pulford and P. 0. P. Ts'o, Biochemistry 19, 4688 (1980). 20. D . M. Cheng, L.-S. Kan, E. E. Leutzinger, K. Jayaraman, P. S. Miller and P. 0. P. Ts'o, Biochemistry 21, 621 (1982). 21. D. M. Cheng, L.-S. Kan, V. L. h o m o and P. 0. P. Ts'o, Biopolymers 23, 575 (1984). 22. D. M. Cheng, L.-S. Kan, D. Frechet, P. 0. P. Ts'o, S. Uesugi, T. Shida and M. Ikehara, Biopolymers 23, 775 (1984). 23. F. Hillenkamp, in Secondary Ion Mass Spectrometry (SlMS V), ed. by A. Benninghoven, R. J. Colton, D. S. Simons, H. W. Werner, Springer Verlag, Berlin 1985, p. 471-475.
Received 16 January 1990; accepted 20 February 1990.
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Lou-Sing Kan Department of Biochemistry, Division of Biophysics, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA
A Wiley-McLaren type time-of-flight mass spectrometer has been used for molecular weight measurements of several unprotected oligodeoxyribonucleotides using matrix-assisted UV laser desorption. Approximately 10 to 100 pmol of sample was required for recording their positive-ion mass spectra with a mass resolution in the range of 150 to 300 (Full width at half maximum) (FWHM). Little fragmentation was observed.
Ultraviolet laser-desorption mass spectrometry (UVLD-MS) has become increasingly important as a method for the sensitive analysis of large bio-organic substances following the development of the UV-absorbing matrix-assisted technique. This technique, originally used for determining the molecular weights of has been reported to be applicable to the analysis of polynucleotides as well." Recently, we developed a UVLD-MS instrument' which employs a Wiley-McLarenll type time-of-flight mass analyzer. That instrument was used for molecular weight measurements of proteins up to more than 100 kDa; and in this paper we have utilized the same instrument for the analysis of several oligodeoxyribonucleotides. This technique shows some interesting differences between the matrix-assisted UVLD-MS of oligonucleotides and proteins, and is different as well from oligonucleotide analysis with other desorption techniques. While the mass spectrometric analysis of oligonucleotides using a variety of soft ionization techniques has been reported, they present an interesting analytical challenge because of the presence of multiple negative charges on the connecting phosphate groups. Thus, they are most easily analyzed from the negative-ion mass spectra of derivatized or (in the case of synthetic oligonucleotides) as fully protected samples. McNeal et a/.'*,l3 employed negative-ion plasma desorption mass spectrometry (PD-MS) for the analysis of fully protected oligonucleotides up to the heptamer, while more recently, oligonucleotides with up to 9 protected units have been detected by PD-MS in the positive-ion mode.I4 In the latter study, the amount of analyte consumed after recovery of the analyzed sample was about 1.4 nmol. Negative-ion fast-atom bombardment (FAB) mass spectral analyses of both protectkd and unprotected oligonucleotides have been demonstrated . ' ~ recently with sensitivities of about 10 n m ~ l . ' ~Most ion spray analysis of oligonucleotides in the negativeion mode with a sensitivity of 200pmol has been reported." 'Author to whom correspondence should be addressed.
AI mirror
Quantel
YG 660 quartz lens &$ .. I
I
I sample probe
,drift tube
1
n
detector
'
I PC-AT I
data system
Figure 1. Schematic diagram of the UV-laser desorption/ionization mass spectrometer.
The positive-ion matrix assisted UV laser-desorption mass spectra of peptides and proteins are characterized by singly-charged (protonated) molecular ions, in contrast to the multiply-charged ions of electrospray spectra and the cationized (i.e., MNa') species frequently observed in FAB- and PD-MS spectra. In addition to the efficient interaction of the UV laser irradiation with the highly abundant matrix molecules (generally, nicotinic acid), the resulting electronically excited matrix molecules and ions may also serve as a rich source of protons, which may be transferred to relatively acidic molecules. Thus, our approach was to examine the positive-ion matrix-assisted UV laser-desorption mass spectra of synthetic oligonucleotides. In general, the matrix technique works best when the analyte shows vanishing spectral absorption at the wavelength used for excitation of the matrix. However, as can be seen from the data presented here, nucleotides produce good results as well, despite their relatively high absorptivity at 266 nm. EXPERIMENTAL Mass spectra were obtained on a modified CVC Products (Rochester, NY, USA) model 2000 time-offlight mass spectrometer, described in detail This instrument is shown schematically in Fig. 1. A Quantel (Santa Clara, CA, USA) model YG-660
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Heyden & Son Limited. 1990
RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 4, NO. 4. 1990 99
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DETERMINATION OF OLIGODEOXYRIBONUCLEOTIDES BY UVLD-MS
frequency-quadrupoled Nd:YAG laser (266 nm, 5 ns pulse' duration) has been used for ion desorption. The laser beam has been focused onto the sample surface by a quartz lens of 300 mm focal length to a spot diameter of about 150 pm. In contrast to other laser-desorption mass spectrometer^',^ used for matrix-assisted UVLD-MS, desorbed ions were not accelerated promptly, but were extracted after a suitable delay of several microseconds to allow time focusing. Ions are post-accelerated into a venetian-blind type conversion dynode, with secondary electrons detected by a Thorn/EMI (Fairfield, NJ, USA) electron multiplier. Mass spectra were recorded and stored in a LeCroy (Spring Valley, NY, USA) model 9400A digital oscilloscope/transient recorder and signal-averaged by PC-based software. All the oligodeoxynucleotides used in this report were synthesized using phosphite-triester chemistry'' for previous nuclear magnetic resonance (NMR) studies.2"-22The sodium counter-ion was replaced with ammonium using a D E A E column. Oligonucleotides were prepared for mass spectrometric analysis by dissolving the samples in ultra-pure water. Useful concentrations were found in the range of to M. An aqueous nicotinic acid solution of 5x molar concentration was used. 1 pL of analyte solution and 1pL of matrix solution were mixed on a silver probe tip and blow dried. RESULTS AND DISCUSSION Eight oligodeoxyribonucleotides, containing between 3 and 8 nucleotide units were investigated by matrixassisted UV laser-desorptionlionization mass spectrometry. Their general structure is shown in Fig. 2 and does not include a phosphate group at the 5' end. The oligonucleotides studied included A-A-G (mol.wt [free acid] 893), T-G-G (mol.wt 900), C-C-A-A (mol.wt 1142), C-G-C-G (mol.wt 1174), T-T-G-G (mol.wt 1204), C-C-A-A-T (mol.wt 1446), C-T-T-G-G (mol.wt
0 0
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Figure 2. General structure of synthetic oligodeoxyribonucleotides used in this study.
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Figure 3. Matrix-assisted UV-laser desorption mass spectrum of 40 pmol of the oligodeoxyribonucleotide T-T-G-G (mol.wt 1204). The molar ratio of analyte to matrix was 1:1250. [Calculated MH' = 1204.2 (monoisotopic mass) 1204.8 (average mass)]. The molecular ion intensity is approximately 8% of the base peak at m / z 203 originating from the nicotinic acid matrix.
1493) and T-C-T-T-G-G (mol.wt 1797). All samples examined showed intense signals for the protonated parent molecule. For comparison, non-matrix-assisted UV laser-desorption spectra were obtained for several oligodeoxyribonucleotides, by drying the analyte solution onto a silver substrate without addition of matrix. This method, known to give good results for various types of compounds in the mass range below 2000 Da,23 did not provide significant ion signals in the case of the oligonucleotides. Mass calibration was carrried out using known mass peaks from the nicotinic acid matrix. The observed molecular weights of the oligonucleotides in all cases were found to be within +1 mass unit of their calculated monoisotopic molecular weights. Figure 3 shows the matrix-assisted UV laserdesorption mass spectrum of the oligodeoxyribonucleotide T-G-G-G. 1.OpL of a 4 x 1 0 - ' ~solution mixed with 1 pL of the nicotinic acid solution was used, corresponding to 40pmol of sample and a molar ratio of sample to matrix of 1:1250. The mass spectrum reveals several differences from those of proteins obtained on the same instrument with the same preparation technique.' First of all, matrix-specific ions of high intensity are observed. In addition, the quasimolecularion region shows strong asymmetric peak broadening above the mass of the protonated molecule. While such broadening might be attributed in part to the presence of unresolved adduct ions, it is noteworthy that lowering the potential of the post-accelerating dynode reduces the effect. As noted before, nucleotides have a strong absorption at 266 nm, whereas peptides show only minor spectral absorption at this wavelength. Peak broadening may thus be due to decay of internally excited metastable ions after acceleration and prior to post-accceleration. The electron multiplier would then detect neutral fragments of decayed parent molecules at a later time than post-accelerated quasimolecular ions. Using a wavelength off the absorption band of nucleotides, such as the frequency tripled Nd:YAG
100 RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 4, NO 4. 1990
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wavelength at 355 nm, in combination with a suitable matrix for this wavelength should lead to a lower internal excitation of the nucleotide molecules and thus to a lower degree of metastable-ion decay. Except for the losses of a guanine base for T-G-G and T-T-G-G, fragment ions were not observed in the matrix-assisted UV-laser desorption mass spectra of oligonucleotides. All other signals observed below the quasimolecular-ion regions are due to known photochemical oligomers and fragments of the nicotinic acid matrix. The mass resolution in matrix-assisted UV-laser desorption of high-mass molecules is known to be limited by the formation of unresolved adduct ions and other specific processes, rather than the inherent resolution of the i n ~ t r u m e n tIn . ~ contrast to recent analyses of large proteins with masses up to 118 000 Da, done on the identical instrument,' the mass resolving power for oligodeoxyribonucleotides of masses below 2000 Da was as high as observed for infra red (IR) laser-desorption18 and non-matrix-assisted UV laserdesorption, i.e., in the range of 150 to 300 measured as full width at half maximum (FWHM). The instrumental mass resolving power is high enough to resolve protonated, cationized and matrix-attached molecules in this mass range. In contrast to the analysis of proteins, intense signals of matrix-attached analyte ions were not observed. In addition to the protonated parent molecule, the most prominent quasimolecular ions observed (in a few cases) were those correspnding to attachment of ammonium or alkali ions. Figure 4 shows the molecular-ion region of the oligodeoxyribonucleotide C-C-A-A (mol.wt 1142), in which the protonated molecular ion is observed with a mass resolving power of about 285, (FWHM). Figure 5 shows the mass spectrum of C-G-C-G (mol.wt 1174), for which a strong peak corresponding to the MNH: ion is also observed. Since attachment of the parent molecule by matrix molecules or cations affects the mass resolution (and mass accuracy) when high-mass peaks are not resolved, the lack of attachment of nicotinic acid to 100
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Figure 5. Matrix-assisted UV-laser desorption mass spectrum of 100 pmol of the oligodeoxyribonucleotide C-G-C-G (mol.wt 1174). The molar ratio of analyte to matrix was 1500. [Calculated MH' = 1174.2 (monoisotopic mass), 1174.2 (average mass)].
oligonucleotides suggests, that analysis of larger polynucleotides might lead to higher effective mass resolution, compared to protein analysis, where attachment by nicotinic acid is ~ o m m o n . ~ , ~ In contrast to the other samples tested, C-G-C-G is a self-complementary oligonucleotide, capable of forming double-stranded molecules with itself (duplexes) at room temperature in 0.1 M neutral aqueous solution. However, the intensity of the [2M+H]+ ion was not higher than for the other oligonucleotides, indicating that duplexes of C-G-C-G have either not been desorbed intact or their formation has been disturbed by the highly abundant nicotinic acid. The formation of oligomeric ions of the parent molecules is, however, strongly affected by the analyte concentration in the analyte/matrix mixture, as observed for peptides as well. Figure 6 shows a spectrum of T-T-G-G for which 100
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Figure 4. Matrix-assisted UV-laser desorption mass spectrum of 100 pmol of the oligodeoxyribonucleotide C-C-A-A (mol.wt 1142). The molar ratio of analyte to matrix was 1:500. [Calculated MH+ = 1142.2 (monoisotpic mass), 1142 (average mass)].
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DETERMINATION O F OLIGODEOXYRIBONUCLEOTIDES BY UVLD-MS
the concentration of the analyte in this case was 1.4 x ' ~ O - ~molar corresponding to 200 pmol of analyte and a molar ratio of analyte to matrix of only 1:350. The intensity of dimeric (duplex) ions is much higher compared to the value in the spectrum in Fig. 3, and the peaks ai-e much broader. Samples with analyte molar resulted in concentrations of more than only weak signals of the analyte, comparable to those in the spectra taken with non-matrix-assisted .UV laser desorption. Conclusions Positive-ion matrix-assisted UV laser-desorption provides an easy, fast and sensitive method of moleular weight determination of synthetic, unprotected oligonucleotides. Derivatization was not necessary to compensate for the large number of negative charges. Positively charged ions were detected easily, despite the highly anionic character of the analyte and in contrast to analysis by other mass spectrometric iondesorption techniques. Acknowledgements This work was supported by grants DIR 86-10589 from the National Science Foundation and GM 34252 (to LSK) from the National Institutes of Health. B. Spengler was supported by a Deutsche Forschungsgemeinschaft Fellowship. We also thank A. Ono for technical assistance. Mass spectral analyses were carried out at the Middle Atlantic Mass Spectrometry Laboratory, an NSF supported Shared Instrumentation Facility. REFERENCES 1. M. Karas and F. Hillenkamp, Anal. Chem. 60, 2299 (1988). 2. M. Karas, U. Bahr, A. Ingendoh and F. Hillenkamp Angew. Ckem. l n f . Ed. En@" 28, 760 (1989). 3. M. Karas, U. Bahr and F. Hillenkamp, Int. J. Mass Spectrom. Ion Proc. 92, 231 (1989).
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Received 16 January 1990; accepted 20 February 1990.
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