Anal. Chem. 1995, 67, 2924-2930
Analysis of Modified Oligonucleotides by Matrix-Assisted Laser Desorption/lonization Fourier Transform Mass Spectrometry E. A. Stemmier* Depatfment of Chemistry, Bowdoin College, Brunswick, Maine 040 11
1111. V. Buchanan, G. B. Hurst, and R. L. Hettich* Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6365
Matrix-assistedlaser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance mass spectrometry (Fl’MS) has been applied to the structural characterization of modified oligodeoxyn’.bonucleotide4-, 6-,and 11-mers. Each oligonucleotidecontained one modified base, either an 06-methyl-substituted guanine, an N6-(1OR)-transopened benzo[alpyrenediol epoxide adduct of adenine, or an W-(R)-styrene oxide adduct of guanine. S-Hydrowpicolinic acid was used as the MALDI matrix for molecular weight and purity determinations, while either 2,5-dihydroxy-bemic acid (DHBA) or an anthradic/nicotinic acid (“A) mixture was used to induce fragmentation for the production of structurally significant fragment ions. For the 4- and 6-mers, the oligonucleotide sequence could be obtained from the direct AA/NA or DHBA spectra. Sequence information was also obtained by inserting a time delay between the laser desorption event and ion detection to permit metastable decomposition. For the 11-mers, high-mass sequence ions were not detected. Although similar sequence ions were observed in both the positive and the negative ion mass spectra, more fragmentation was generally observed in the positive ion mode. In the positive ion mode, modified base fragment ions were observed when DHBA was used, and these fragments were examined using accurate mass measurements, collisionally induced dissociations, and ionmolecule reactions to characterize the modi6ed base. MALDI-FIMS signalsfrom one sample application can be used for the measurement of hundreds of spectra. The direct MALDI-FIr mass spectra show matrix-dependent, structurally informative fragments, and CID experiments can be implemented using low-picomole sample quantities. An important component of research directed at understanding the molecular basis for cancer involves the detection and structural characterization of oligonucleotides modified by interactions with carcinogenic agents.’ In addition to the more challenging task of characterizing oligonucleotides modified by in vivo or in vitro reactions, methods are needed for the characterization of sitespecificallymodified oligonucleotides used in studies of the effects (1) Phillips, D. H. In The Molecular Basis of Cancer; Farmer, P. B., Walker, J. M., Eds.; Wiley-Interscience: New York, 1985; pp 133-179.
2924 Analytical Chemistry, Vol. 67, No. 17, September 1, 7995
of carcinogens on DNA replication and repair.2 Information that is relevant to the structural characterization of modified oligonucleotides includes the determination of the type of modification and identification of the base or nucleotide unit that has been modified. Regarding nucleobase modifications, identification of the specific site that has been modified is generally desired, while information about the oligonucleotide sequence and the site of the modiiied nucleotide unit within the sequence is often of interest to determine the role of adjacent nucleotides on modifications. The application of mass spectrometry to oligonucleotide analysis3,*initially involved techniques such as fast atom bombardment (FAB)5and plasma desorption (PD).6 FAB7and FAB with collision-induced dissociations (CID or MS/MS)*have been used to identify the position of a modification in the sequence of modified oligonucleotides. In general, the poor sensitivities associated with FAB and PD (requiring nanomolar quantities of underivatized material) and problems with spectral reproducibility and chemical noise have limited the application of these techniques to oligonucleotide analysis. In recent years, two mass spectrometric ionization techniques, matrix-assisted laser desorption/ionization (MALDD9 and elec(2) Basu. A. IC;Essigmann. J. M. Chem. Res. Toricol. 1988,1, 1-18. (3) McCloskey, J. A.; Crain, P. F. Int. J. Muss Spectrom. Ion Processes 1992, 118/11g3 593-615. (4) Crain, P.F. Muss Spectrom. Rev. 1990,9, 505-554. (5) (a) Grotjahn, L.; Frank, R; Blocker, H. Nucleic Acids Res. 1982,10,46714678. @) Grotjahn. L.: Blocker, H.; Frank, R Biomed. Muss Spectrom. 1985, 12,514-524. (c) Liguori, A.; Sindona, G.; Uccella, N. Biomed. Environ. Muss Spectrom. 1988,16,451-454. (d) Cemy, R. L.;Tomer, K. B.; Gross, M. L.; Grotjahn, L. Anal. Biochem. 1987,165, 175-182. (6) (a) McNeal, C. J.; Ogilvie, K. K.; Theriault, N. Y.: Nemer, M. J. J. Am. Chem. SOC.1982,104, 976-980. @) Viari, A,; Ballini, J.-P.; Vigny, P.; Shire, D.; Dousset, P. Biomed. Environ. Mass Spectrom. 1987.14, 83-90. (c) Viari, A.: Ballini. J. P.; Meleard, P.; Vigny. P.; Dousset, P.; Blonski, C.; Shire, D. Biomed. Environ. Mass Spectrom. 1988,16, 225-228. (7) Iden, C. R; Rieger, R. A. Biomed. Environ. Muss Spectrom. 1989,18,617619. (8) Dino, J. J., Jr.; Guenat, C. R; Tomer, IC B.: Kaufman, D. G. Rapid Commun. Mass Spectrom. 1987,1. 69-71. (9) (a) Nordhoff, E.; Ingendoh, A; Cramer, R.; Overberg, A; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun. Mass Spectrom. 1992,6 , 771-776. @) Wu, K. J.; Steding, A; Becker, C. H. Rapid Commun. Muss Spectrom. 1993,7, 142-146. (c) Nordhoff, E.; Karas. M.; Hahner. S.; Hillenkamp, F.; Kirpekar, F.; Kristiansen, IC;Roepstorf[, R. Proc. 4FdASMS Conf on Muss Spectrom. and Allied Topics, Chicago, IL. May 29-June 3. 1994; p 971. (d) Parr, G. R ; Fitzgerald, M. C.; Smith, L. M. Rapid Commun. Muss Spectrom. 1992,6.369-372. (e)Schneider, K; Chait. B. T. Org, Muss Spectrom. 1993,28. 1353-1361. 0003-270019510367-2924$9.00/0 0 1995 American Chemical Society
trospray (ES),l0 have been successfully applied to the sensitive detection of oligonucleotides. MALDI time-of-flight (TOF) mass spectrometry is a sensitive technique that has been applied to the analysis of chemically m o d ~ e doligonucleotides," including oligonucleotides containing modified bases such as uracil glycol, bromoguanine, or O6-buty1guanine.llb The low-mass resolution and the inability to conduct MS/MS experiments on typical TOF systems limits the amount of structural information for oligonucleotides that may be obtained from MALDI-TOFexperiments. In contrast, Fourier transform ion cyclotron resonance mass spectrometry (FTMS) with MALDP has the potential to provide detailed structural information for biomolecules using the highresolution, accurate mass, and ion trapping capabilities of FTMS. Recent experiments have demonstrated the measurement of highand ultrahigh-resolution mass spectra of proteins and peptides.lZc Past studies from our laboratory have shown that MALDIFTMS can be used for the structural characterization of normal and modified nucleic acid components.13 In this report, MALDIFTMS at 355 nm is applied to the structural characterization of modified oligonucleotide 4,&, and 11-mers with the base modifications shown in Figure 1. Experimental and mass spectral considerations that influence selection of an appropriate MALDI matrix are addressed, and features of the positive and negative ion MALDI spectra are examined. The ability of MALDI-FTMS to determine molecular weight and base sequence information and to provide detailed structural characterization of the nucleic base modification has been assessed.
(1) 5'-TGMeCA-3' (2) 5'-GCTAGMeC-3'
(3) S - A ~ ~ ~ C G A G G - Y (4) 5'-CGGTCABP'CGAGG-3' (5) 5'-CGGACABP2AGAAG-3
(6) 5'-GGCAGGStY'TGGTG-3' 0
I
-- ABPl
- ABPZ
-
Figure 1. Structures of the modified oligonucleotides 1-6.
translational energy range) into the argon collision gas at a static pressure of -(2-3) x Torr. H/D exchange reactions were carried out by isolating the ion of interest in the presence of 1.2 x 10+ TOITof DzO. Adducts and Sample Preparation. The TGveCA (1) and EXPERIMENTAL SECTION Instrumentation. An Extrel FTMS2000 system (Extrel GCTAGMeC(2) (GMe= 06-methylguanine) samples were obtained FTMS, Madison, WI) equipped with a 3-T superconducting from Dr. John M. Essigmann (Department of Applied Biological magnet and a differentially pumped dual ion cell was used for Sciences, Massachusetts Institute of Technology) .15 The MALDI experiments. An ion deceleration technique, first deABPICGAGG(3) and CGGTCABPICGAGG(4), where ABp' is scribed by CastorolZband adapted for our 3-T FTMS ~ y s t e m , ~ ~(7R,8S,9S,lOR)-*-[ ~~~ 10-(7,8,9-trihydroxy-7,8,9,1C-tetrahydrobenzowas used for all experiments. Source side detection was used [alpyrenyl)ladenine, samples were obtained from Dr. Donald following wide-band stored-waveform inverse Fourier transform14 Jerina (National Institutes of Health).16 The CGGACABPZAGAAG (SWIFI? excitation. Typically, 25 single-shot spectra were aver(5),where ABp2is (7S,8R,9S,lOR)-P-[10-(7,8,9-trihydroxy-7,8,9,1@ aged to improve the signal-to-noise ratio, although complete tetrahydrobenzo [alpyrenylladenine, and GGCAGGSW'GGTG(6), spectra could be measured with a single laser shot. CID where GsW is NZ-(8-styrene oxide guanine, samples were obtained experiments were performed by isolating the ion of interest using from Drs. Constance and Tom Harris (Chemistry Department, a SWIFT waveform, followed by acceleration (100-500 eV Vanderbilt University) . I 7 The aqueous sample solutions (W4M) were prepared for MALDI analysis by mixing the matrix (10) (a) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Chem. SOC.1993,115, solution (1-5 pL) with the sample solution (1 pL) to obtain a 12085-12095. (b) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. matrix-to-analyte mole ratio in the range of 104:1to 103:l. One 60-70. (c) Little, D. P.; Chorush, R A,; Speir, SOC.Mass Spectrom. 1992,3, J. P.; Senko, M. W.; Kelleher, H. L.; McLafferty, F. W. J. Am. Chem. SOC. microliter of this mixture containing 15-65 pmol of analyte was 1994,116, 4893-4897. (d) Reddy, D. M.; Rieger, R. A,; Torres, M. C.; applied to the probe following the application of a suspension of (e) McLuckey, S. A; HabibiIden, C. R Anal. Biochem. 1994,220,200-207. ammoniumactivated ion-exchange beads (Dowex 5OW-Xl2,lOOGoudarzi, S. J. Am. Sot. Mass Spectrom. 1994,5, 740-747. (11) (a) Costello, C. E.; Nordhoff, E.; Hillenkamp, F. Int. J Mass Spectrom. Ion 200 mesh; Bio-Rad Laboratories, Richmond, CA). The analyte/ Processes 1994,132,239-249. (b) Wang, B. H.: Biemann, K Anal. Chem. matrix mixture was applied to the probe tip, resulting in surface 1994,66, 1918-1924. (c) Hathaway, G. BioTechniques 1994,17, 150coverage of -7 f 2 mm2 for a 1-pL sample application, and 155. (d) Pieles, U.; Ziicher. W.; Schar, M.; Moser, H. E. Nucleic Acids Res. 1993,21, 3191-3196. samples were air-dried without assistance. Matrix materials were (12) (a) Buchanan, M. V.; Hettich, R. L. Anal. Chem. 1993,65,245-259A (b) purchased from Aldrich Chemical Co. (Milwaukee, WI) and were Castoro, J. A.; Koster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6, 239-241. (c) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993,65, 26212627. (13) (a) Hettich, R L.; Buchanan, M. V. Int.J Mass Spectrom. Ion Processes 1991, 111, 365-380. (b) Hettich, R.; Buchanan, M. ]. Am. SOC.Mass Spectrom. 1991,2, 402-412. (c) Stemmler, E. A,: Buchanan, M. V.; Hurst, G. B.; Hettich. R. L. Anal. Chem. 1994,66, 1274-1285. (d) Stemmler, E. A;
Hettich, R. L.; Hurst, G. B.; Buchanan. M. V. Rapid Commun. Mass Spectrom. 1993,7, 828-836. (e) Nourse, B. D.; Hettich, R. L.; Buchanan, M. V. J Am. SOC.Mass Spectrom. 1993,4, 296-305. (14) Chen, L.: Wang, T.-C. L.; Ricca, T. L.; Marshall, A G. Anal. Chem. 1987. 59.449-454.
(15) (a) Essigmann, J. M.; Fowler, K W.; Green, C. L.; Loechler, E. L. Enuiron. Health Penpect. 1985,62. 171-176. (b) Essigmann, J. M.; Loechler, E. L.; Green, C. L. Prog. Clin. Biol. Res. 1986,209A. 433-440. (16) (a) Lakshman, M. K; Sayer, J. M.; Jerina, D. M.]. Am. Chem. SOC.1991, 113,6589-6594. (b) Lakshman, M. K; Sayer, J. M.; Yagi, H.; Jerina, D. M. J. Org. Chem. 1992,57, 4585-4590. (c) Schurter, E. J.; Sayer, J. M.; Oh-
hara. T.: Yagi, H.; Jerina, D. M.; Gorenstein. D. G., manuscript in preparation. (17) (a) Kim, S. J.; Stone, M. P.; Harris, C. M.; Harris, T. M. J. Am. Chem. SOC. 1992,114, 5480. (b) Harris, T. M.; Harris, C. M.: Kim, S. J.; Han, S.; Kim, H. Y.Zhou. L. Polycyclic Arom. Hydrocarbons 1994,6, 9-16.
Analytical Chemistiy, Vol. 67, No. 17, September 1, 1995
2925
I
CGGTCA~~~CGAGG (4)
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Figure 2. Negative ion MALDI-FT mass spectra obtained with HPA as the matrix. (A) TGMeCA(1): M, = 1187.86;>30 pmol applied, 10 0OO:l matrixianalyte mole ratio, inset shows a high-resolution measurement (range of m/z 1180-1 194).(6)GCTAGMeC(2):Mr = 1806.25,260 pmol applied, 5000:l matrixianalyte mole ratio, inset shows a high-resolution measurement (range of mlz 1795-1815).(C) ABPICGAGG (3): Mr = 2143.59,60 pmol applied, 7000:l matrixianalyte mole ratio. (D) CGGTCABPICGAGG (4):Mr = 3684.6,measured mass = 3684.3,65 pmol applied, 5000:l matrixianalyte mole ratio. (E) CGGACABP2AGAAG(5): M, = 3701.61,measured mass = 3701.1,15 pmol applied, 30 0OO:l matrixianalyte mole ratio. (F) GGCAGGstyTGGTG (6):Mr = 3574.44,measured mass = 3573.8,20 pmol applied, 20 0OO:l matrixianalyte mole ratio.
used as received without further purification. Matrix solution concentrations, 1.5M for 2,5dihydroxybenzoic acid @HBA) and 0.5 M for 3-hydroxypicolinic acid (HPA), were close to the solubility limit for each compound in 1:l CH&N/H20. The anthranilic acid (AA)-nicotinic acid (NA) matrix mixture contained a 4:l mole ratio of the two matrices, with a total concentration of 0.3 M. RESULTS AND DISCUSSION
The mass spectral characterization of the oligonucleotides shown in Figure 1 has been divided into three main areas: (1) molecular weight and purity determinations, (2) sequence ions, and (3) modified base characterization. The successful application of MALDI-FTMSto the analysis of oligonucleotides has required the combined use of an appropriate MALDI matrix with ion deceleration techniques. The ion deceleration technique and matrix effects, as applied to the analysis of oligonucleotides on our 3-T FTMS system, have been d e ~ c r i b e d ; ' ~and ~ , ~three matrices, HPA, DHBA, and AA/NA, were used in this study. We have not evaluated the ultimate sensitivity of MALDI-FTMS for oligonucleotide analysis; however, the quantities used in this study (15-65 pmol applied) are significantly lower than sample quantities required for FAB analysis of underivatized materials. Molecular Weight and Purity Determinations. For the MALDI-FTMS analysis of oligonucleotides, HPA is the matrix of choice for molecular weight and purity determinations. When HPA is used, minimal oligonucleotide fragmentation is observed in both negative and positive ion mass spectra. Low-resolution negative ion mass spectra of the modified oligonucleotides 2926
Analytical Chemistty, Vol. 67, No. 17, September 1, 1995
examined in this study are shown in Figure 2, and [M - HIions are observed for all the modAed oligomers. With the exception of the BPDEmodified hexamer 3, the only fragment ions detected result from loss of neutral guanine (HG) for the 11-mers 4 and 6. Observation of HG loss has also been reported in the HPA MALDI-ETmass spectrum of a mixed-base 12-mere13d The spectrum of the BPDE-modilied hexamer 3 shows a few fragment ions, primarily an ion resulting from cleavage of the phosphodiester bond to give a Y3- ion (described below). No evidence for sample impurities was found. The spectra shown in Figure 2 were measured under lowresolution conditions (acquisition times of -30 ms). Under these conditions, the resolving power for the [M - HI- ions from the three 11-mers is in the range of 175-325. Improvements in resolving power depend upon the generation of a population of long-lived ions, where ion lifetime may be limited by collisions or metastable decay. The measurement of high- and ultrahighresolution mass spectra by MALDI-FTMShas been reported for peptides and small proteins when steps, such as the addition of h c t o s e to the DHBA matrix and the careful control of laser irradiance, have been taken to reduce metastable decay of the MALDI-desorbed ions.lZc From our work, metastable decay appears to be a more significant problem for the MALDI-FTMS analysis of oligonucleotides than for proteins. For example, when a 100-500 ms delay is inserted prior to detection of the modified hexamers 2 and 3,metastable decay of the deprotonated molecular ion becomes apparent (see Figure 3). Decay rates appear to increase with the size of the oligomer and are strongly influenced
1
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Figure 3. Negative ion MALDI-FT mass spectra measured with HPA as the matrix. (A) GCTAGMeC(2) measured with a 100-ms delay inserted prior to ion detection and (e) ABPICGAGG (3) measured yith a 500-ms delay inserted prior to ion detection.
by the MALDI matrix and base composition. At present, resolving powers of 3700 and 1700 have been obtained for the modified tetramer 1 and hexamer 2, respectively (see insets in Figure 2). Studies directed at improving the high-resolution capabilities of FTMS for the analysis of oligonucleotides are under investigation. The measured isotopic distribution for the [M - HI- ion from 1 (shown in the inset in Figure 2A) is skewed relative to that expected from 13C contributions. This effect becomes more pronounced when DHBA is used as the MALDI matrix. This distortion of the measured isotopic distribution, attributed to hydrogen additions, has been observed in previous MALDI-FTMS studies'3b.c in which DHBA was used as the matrix. Similar additionshave been reported in FAB studies of oligonucleotides'a and attributed to hydrogen radical additions or abstractions involving the matrix or the reduction of protonated ions. The low-resolution spectra shown in Figure 2 were calibrated using an external calibration mixture of oligopolythymidylic acids (pd06, pdOs, and pdmlo), whose spectra were measured using the same matrix and ion deceleration conditions. For the lowresolution measurements of the three 11-mer samples 4-6, calibrations using relative molar masses for the calibrants were applied. External calibration gave measured masses with an average deviation from the calculated mass equal to -0.5 Da. In general, mass accuracy and resolving power are significantly higher for smaller oligomers or for oligonucleotide fragments produced when matrices such as DHBA are used (see below). Sequence Ions. In addition to molecular weight, mass spectral determination of the base sequence of the modified oligonucleotide is highly desirable. Ideally, the direct or MS/ MS mass spectrum should provide the information necessary to determine the base sequence to eliminate the use of timeconsuming enzymatic or chemical degradation of the sample. We have found that the DHBA MALDI-FTmass spectra of oligomers up to tetramers are generally dominated by Y- and ions 80 Da higher in mass than X series ions, where Y series ions result from cleavage at the 3' carbon and X series ions result from cleavage at the 5' carbon6b (see Scheme 1). The X and Y ion series (18) (a) Grotjahn. L. Springer Proc. Phys. 1986, 9, 118-125. @) Cerny, R L.; Gross, M. L. Anal. Chem. 1 9 8 5 , 57, 1160-1163. (c) Laramee, J. A; Arbogast, B.:Deinzer, M. L. Anal. Chem. 1 9 8 9 , 61, 2154-2160.
.... ISOB
Y- ions
correspond to w- and d-type ions, respectively, using the nomenclature proposed by Mchckey et al.lobWe have determined that the [X 801- ions are X ions associated with a reduced sugar ring ([X C&O]- ions) on the basis of accurate mass measure ments. X-type fragment ions are found at significantly lower abundance, if they are observed at all. When X-type ions are observed, they are generally accompanied by the more abundant [X C5H40]- ions. The positive and negative ion MALDI-FT mass spectra show similar fragment ions, with positive ions appearing at Y H 2 + and [ X H 2 C&OI+. In general, molecular ion signals are weaker and low-mass, baserelated ions are more abundant in the positive ion mode. The formation of Y- and [X CsH401- ions in the MALDI-FT mass spectra can be rationalized by assuming that fragmentation is initiated by loss of a neutral base, followed by cleavage at the 3' carbon. This process is depicted for TGMeCA(1) in Scheme 2, which shows fragmentation initiated by loss of the modified guanine, HGMe. Depending on where the charge resides, Yz-or [XI+ CsH401- ions are produced in this example. Similar nucleobase loss-initiated fragmentations have been proposed in previous ESoa and MALDI-TOP studies. For the MALDI-FT mass spectra, neutral base loss-initiated cleavages also can be used to rationalize the production of sequencerelated fragments that are observed in the positive ion mass spectra. While the details of this fragmentationmechanism are still under investigation, the route shown in Scheme 2 serves to rationalize the negative and positive ion spectra measured in our study. T W C A Sequence Zons. The negative ion DHBA MALDI-FT mass spectrum of TGMeCAis shown in Figure 4A. To identify sequence-related ions in the negative and positive ion spectra of TGMeCA,mass spectral windows were examined where YI-, XI-, or [X,, + CsH401- fragments (and methylated analogues 14 Da higher in mass) were expected. The subscripts are used to indicate the cleavage point in the sequence, with bases numbered beginning at the 5' end for X ions. Masses for the YE-, X,-, and [XI + CsH401- fragments are shown in Scheme 1. As described above, X-type ions have always been found to exhibit low abundance in the 355nm MALDI mass spectra, and ions found
+ +
+
+
+
Analytical Chemistty, Vol. 67, No. 17, September 1, 1995
2927
Scheme 2
r
L
0
OH
- ; ? O w OH
OH
[M-H]-
[M-H-HG~T-
yp'
OH
m/z 619 reported in IR MALDI-TOF studies, where intense IR radiation is used to induce prompt oligonucleotide fragmentation.gc In the positive ion mode, the YIHzt and YzHzt ions are observed at mlz 332 and 621. Based on detection of the Y-type ions alone, the sequence and site of modification can be determined. In addition, the spectra show a modified [XZ CsH401- ion at mlz 744 and a modified [X, CsH401- ion at mlz 1033, and these ions appear 2 Da higher in mass in the positive ion mass spec!". Detection of these fragments provides further confirmation that a modifed guanine residue appears at position 2 in the sequence, based on sequencing from the 5' end of the Cmer. GCT.AWeC Sequence Ions. The negative ion mass spectrum of GCIAW'C (2), measured using AA/NA as the MALDI matrix, is shown in Figure 4B. To determine the base sequence and the identity of the modified base, the AA/NA MALDI-IT mass spectra were compared to the HPA mass spectra measured with a delay inserted between desorption and ion detection (see Figure 3A). Masses for the Yn-, Xn-, and [X, + C5H401- fragments are shown in Scheme 1. In mass spectral windows where these ions were expected, abundant Y5-, Y4-, and Yz- ions were detected. Very weak signals were detected for Y1- and Y3-. All of the detected Y-type ions, with the exception of Y1-, appear 14 Da higher in mass, indicating that the site of modification was base 5 at the 3' end of the hemmer. Thus, the base sequence and the site of the modified base can be determined on the basis of detection of the Y-type ions. The very weak Y3- ion would probably have gone undetected in the analysis of an unknown sequence; however, additional information is available from [X, + CsH401- ions. The [X, + CsH401- ion at m / z 1019 can be used to determine that the sequence is GCTAGMeC,not GCATGMeC,and the presence of T at position 3 in the sequence is consistent with the absence of Y3- and [X:! + C~H401-ions. ABPTGAGG Sequence Ions. The DHBA MALDI-FT mass spectrum of ABPICGAGGis shown in Figure 4C, and the expected Yn-, X,,-, and [X, + CsH401- fragments are shown in Scheme 1. The full range of Y-type ions is found by examining the negative ion DHBA mass spectra and the negative ion HPA MALDI mass spectrum measured with a delay prior to ion detection (see Figure 3B). The Yl--Y5- sequence ions, detected at mlz 346,675,988, 1317, and 1606, show no evidence for nucleobase modification. From these fragments, the sequence is coniirmed and the site of
+
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Figure 4. (A) Negative ion MALDI-FT mass spectrum of TG'eCA (1) measured with DHBA as the matrix. (B) Negative ion MALDI-FT mass spectrum of GCTAGMeC (2) measured with ANNA as the matrix. (C) Negative ion MALDI-FT mass spectrum 3f AaPICGAGG (3) measured with DHBA as the matrix.
within the windows for Yn- and Xn- ion were assigned as Y-type ions. The negative ion spectrum of 1shows unmodified Y1- and Yz- ions at m / z 330 and 619, with a weak modifed Y3- ion at mlz 962. The Y1- ion, which appears as a weak peak in the spectrum shown in Figure 4A, is more abundant in negative ion spectra measured with shorter ion deceleration times. In the spectral window where Y3- and X,- ions are expected, two ions of comparable abundance were detected at m / z 953 and 962. The mlz 953 fragment was determined to be the X,- fragment on the basis of the detection of an [X,+ CsH401- ion 80 Da higher in mass at m / z 1033. The Y3- ion is thought to be weak because ion formation is initiated by the unfavorable loss of HT. The unfavorable loss of HT leading to weak Y-type ions has been 2928 Analytical Chemistty, Vol. 67,No. 17, September 1 , 1995
+
modification may be assigned as the 5' end adenine in the sequence. The identification of adenine as the modified base can be confirmed by losses from [M - HI- shown in F i e 3B, where the loss of 437 Da corresponds to the loss of HABp1. In addition, confirmation of the modifled base may be obtained from the lowmass positive ion mass spectrum, as described below. At present, the DHBA or AA/NA MALDI-FT mass spectra, coupled with HPA measurements with time delays to permit decomposition, provide information that can be used to partially or totally confirm the sequence of small oligonucleotides. For the larger oligonucleotides, we were only able to confirm the sequence using the ions YI--Y4- because of extensive oligonucleotide fragmentation. Modified Base Characterization. For the structural characterization of modified oligonucleotides, information regarding the base that has been modified and the site of modification is often desired. In the analysis of oligonucleotides with DHBA as the MALDI matrix, the positive ion mass spectra show low-mass, base-related fragment ions that include abundant signals for the protonated bases AH2+, GHz+, and CHz+ for unmodified oligonucleotides. Thymine, which has the lowest proton affinity1gof the four DNA bases, has not been observed to produce the THz+ ion in the DHBA MALDI-FT mass spectra of oligonucleotides. In addition, positively charged [B C5H,jO] fragments containing the bases A, G, and C (represented as SA+,sG+, or sC+) are observed. In the low-mass region of the DHBA W E F T negative ion mass spectra of oligonucleotides larger than dinucleotides, negatively charged base ions have not been detected. In this section, the positively charged fragment ions have been studied using accurate mass, CID, and ion-molecule reactions in order to characterize the three types of base mod~cations examined in this study. 06-MeNtylguanine. In the positive ion DHBA MALDI mass spectra of oligonucleotidesTGM'CA (1)and GCTAWeC (2),lowmass protonated base and [B sugar] fragments are detected (see Figure 5A for 2). In the spectrum of the modified tetramer 1,where the sole guanine residue is modified, the only protonated guanine peak corresponds to the modified base, GMeH2+,at m / z 166. In the case of the modified hexamer, both a modified and an unmodified guanine residue are present, and peaks at m / z 152 and 166, corresponding to both bases, appear in the spectrum (see Figure 5A). Accurate mass measurements support the mass assignments. To verify that the modified base is a guanine methylated at the 06-position CID mass spectra of the GMeH2+peaks were measured. For the 4 and 6mers (1 and 2), CID mass spectra of the GMeH2+peak showed one fragment resulting from the loss of NH3. Experiments using all the methylguanine isomers as precursors to the corresponding WH2+ ions on our FTMS system showed that NH3 loss is a comqon feature of the low-energyCID mass spectra of all protonated methylated guanine isomers, with one exception. The one exception, A"-methylguanine, loses NHzCH3 instead of NH3. Thus, CID spectra of the GMeHz+ion support assignment of the peak as originating from a methylguanine and rule out substitution at the N'-position; however, the spectra do not provide evidence for substitution at the 06-position, and additional experiments are required to provide this information.
+
+
(19) Wilson, M. S.; McCloskey, J. A j . Am. Chem. SOC. 1975, 97, 3436-3444.
1000,
I
1
.A_
100.0,
1
166 G~*H*+
/
00 14500
15000
15500
18000
18500
17000
175W
17000
17500
100 0
EO 0
167
I
00
14500
15000
15500
lB000
16500
mlz
Figure 5. Positive ion MALDI-FT mass spectra of GCTAGMeC(2) measured with DHBA as the matrix (A) with 40-psec deceleration time and ejection of matrix ions at m/z 137 and 273. (6) Ion isolation, Torr. (C) Reaction time = 10 reaction time = 0 s, f 0 , O = 1.2 x s, f 0 , O = 1.2 x Torr.
In addition to CID experiments, ion-molecule reactions are possible on the ETMS. Previous FTMS studies from our laboratory,'% where standards of all the methylguanine isomers were studied, showed that H/D exchange reactions can be used to distinguish sites of methyl group substitution. This differentiation is based on determiniig the number of exchangeable hydrogens and the rate for the H/D exchange reaction. This approach was applied to the oligonucleotide-derived methylguanines by isolating the GMeH2+ion and allowing reaction with 1x Torr of DzO. For the modified GCTAGMeChexamer 2, which produces both modified and unmodified guanine fragments, GMeHz+and GH2+,the reactions of both peaks have been followed simultaneously (see Figure 5, parts B and C). The reaction with GH2+ was followed as a reference to determine relative reaction rates. While the unmodified guanine shows the expected rapid exchange of three H for D, only one H/D exchange reaction was observed for the GMeH2+ion. This narrows the range of possible isomers to include guanine modified at the 06-position or the 3-methyl position. By comparing the rate of formation of the [M Dl+ ion from the GMeH2+ion with the rate of formation from GH2+,it was determined that the oligonucleotidewas modified at the 06-positionof guanine. Given the same reaction time, the m / z 167 peak from the $methyl isomer should have exceeded that of m / z 166. BPDE-ModiFed Adenines. In a previous FTMS study,13bthe MALDI mass spectra of a group of PAH-DE nucleoside and nucleotide adducts were studied. In these studies, the positive ion mass spectrum of 7(R),8(S),9(S)-trihydroxy-lO(R)-(Nz-deoxyguanosyl3'-phosphate)-7,8,9,l@tetrahydrobenzo [alpyrene showed signals correspondingto the protonated, adducted base (GB91z+), along with peaks resulting from one and two neutral H20 losses from GBPH2+.Higher abundance peaks were due to the positively charged PAH tiol, R+, at m/z 303, and fragments at m / z 285 and
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Analytical Chemistry, Vol. 67, No. 17, September 1, 7995
2929
1000
I
20 0
250 0
350 0
mi2
400 0
450 0
ao
MOO
i 100 0
200 0
300 0
mh
400 0
, ,
.,
,I,, MO 0
,
.
. 800 0
Flgure 7. Positive ion MALDI-FT mass spectrum of GGCAGGSVG239 1257
~
measured with DHBA as the matrix with a 20-psec deceleration time.
GTG (6)
adducted guanine, GSVH2+, while the m/z 352 peak is the sugar adducted base peak, [sGS@']+. Exact mass measurements, using the GHz+, SA+, and sG+ peaks as calibrants, give mlz 352.1396 (352.1404, calculated) and mlz 272.1170 (272.1142, calculated). Isolation and CID of the mlz 352 ion result in loss of the styrene adduct, leaving the unmodified base sugar peak, sG+, at mlz 232 as the sole product.
+
Figure 6. (A) Positive ion MALDI-FT mass spectra of ABPICGAGG
(3) measured with DHBA as the matrix with 40-psec deceleration times and isolation of mlz 250-500 with ejection of matrix ions at 273; 90 pmol applied. (6) Positive ion CID mass spectra of the [R H 2 0 - CO]+ ion at mlr 257 from CGGTCABPICGAGG (4); 40 pmol
applied. 257, corresponding to [R - HzOI+ and [R- HzO - COP. Based on the results of that study, we have looked for characteristicions expected to appear in the positive ion MALDI-FTMS spectra of the three BPDEmodified oligonucleotides 3-5. The spectra of all three modified oligomers 3-5 show [R - HzO - COl+ ions at m/z 257. When ion isolation techniques are employed to eject more abundant fragment ions and improve the dynamic range for the detection of lower abundance fragments, all of the expected adducted base and BPDE triol fragments are observed (see Figure 6A for hexamer 3). In this spectrum, the adducted base peaks, ABP1H2+, [ABPIHz - HzOl+,and [ABP1H2- 2HzOI+,appear at mlz 438, 420, and 402, respectively while the BPDE triol fragments appear at m/z 303, 285, and 257. Other data supporting assignment of a BPDE-modified adenine come from measurements made using HPA as the matrix. In the negative ion mode, with a delay inserted prior to ion detection, fragment ions resulting from losses of both the BPDE triol and adducted base (losses of 302 and 437 from [M - HI-) are observed for the modified &mer (see Figure 3B) and 11-mer. The positive ion mass spectrum of the BPDE hexamer 3, measured using HPA with a delay inserted prior to ion detection, shows the protonated adducted base peak at m / z 438. When the positive ion DHBA MALDI spectra are measured under higher resolution conditions, using the A H 2 + , GHz+, and SA+ ions as internal calibrants, good agreement is found between the expected and measured masses for the adducted base and BPDE triol peaks. In addition to accurate mass measurements, CID experiments may be used to contirm the identity of the BPDE triol fragments. For example, the CID mass spectrum of the mlz 257 fragment isolated from the DHBA positive ion mass spectrum of the adducted 11-mer 4 is shown in Figure 6B. The characteristic fragments produced at m / z 239 and 215 have been observed in our previous study13band serve to confirm the assignment of this ion as a BPDE triol fragment. N2-Styrene Oxide Adduct of Guanine. The positive ion DHBA MALDI-FT mass spectrum of the styrene adducted 11-mer 6 is shown in Figure 7. In this spectrum, peaks at mlz 352 and 272 are observed. The m / z 272 peak corresponds to the styrene 2930 Analytical Chemistry, Vol. 67,No. 77, September 1, 7995
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CONCLUSIONS
The structures of modified oligonucleotidescan be characterized by MALDI-ETMS using low-picomole amounts of sample. In the negative and positive ion modes, MALDI matrix selection can be used to control the degree of analyte fragmentation. Using HPA reduces the degree of oligonucleotide fragmentation, particularly in the negative ion mode, and this matrix is used for molecular weight and purity determinations. The AA/NA and DHBA matrices are used to induce more fragmentation and provide information regarding sequence ions. The complete base sequence and the identity and location of the modified base in the sequence could be determined for the modified tetramer 1 and hexamers 2 and 3. Extensive fragmentation of the 11-mers prevented determination of the sequence at the 5' end of the oligomer. In the positive ion mode, low-mass ions corresponding to the bases, AHz', CHz', and GH2+ (modified and unmodified), are observed. These low-mass fragments can be used to determine the site of modification on the base. H/D exchange reactions permitted determination of the 06-positionas the site of guanine modification in oligonucleotides 1 and 2. The BPDE and styrene adducted bases were characterized using exact mass and CID techniques. ACKNOWLEDGMENT
The authors thank Dr. John M. Essigmann, Dr. Donald Jerina, and Drs. Constance and Tom Harris for providing the modified oligonucleotide samples used in this study. Research sponsored jointly by the National Cancer Institute under Interagency Agree ments DOE 0485F053-Al and NCIY01-CP-20512-13, the Office of Health and Environmental Research, U S . Department of Energy under contract DE-AC05.840R21400 with Lockheed Martin Energy Systems, Inc., and the DOE Faculty Research Participation Program under contract DE-AC05760R00033 between the US. Department of Energy and Oak Ridge Institute for Science and Education. Received for review February 27, 1995. Accepted June 12, 1995.@
AC9502054 @
Abstract published in Advance ACS Abstracts, July 15, 1995.
