Ionization Mass Spectrometry for

Jun 19, 2001 - Keough, T.; Shaffer, J. D.; Lacey, M. P.; Riley, T. A.; Marvin, W. B.; Scurria, M. A.; Hasselfield, J. A.; Hesselberth, E. P. Anal. Che...
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Anal. Chem. 2001, 73, 3263-3273

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Locating Abasic Sites and Determining the Rates of Enzymatic Hydrolysis of Model Oligodeoxynucleotides Li-Kang Zhang, Don Rempel, and Michael L. Gross*

Department of Chemistry, Washington University, 1 Brookings Drive, St. Louis, Missouri 63130

A method using a combination of exonuclease enzymatic digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was developed to locate model abasic sites in a series of model 21-base oligodeoxynucleotides in which a nucleobase was replaced by a hydrogen atom. The exonuclease digestion rate, with either snake venom phosphodiesterase (SVP) or bovine spleen phosphodiesterase (BSP), clearly slows as the digestion approaches the abasic sites and stops as it reaches it. An oligodeoxynucleotide containing an abasic site in which OH replaces the nucleobase shows similar results. MALDI mass spectra taken at appropriate times during the course of hydrolysis are the basis for rate measurements, and the kinetics also reveal the location of the abasic site. A mathematical treatment of the timedependent MALDI data was implemented to obtain rate curves and rate constants for the enzymatic digestion of both modified and unmodified oligodeoxynucleotides. Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) have moved mass spectrometry (MS) into position to determine rapidly the sequence of oligodeoxynucleotides.1-4 The use of MALDI to analyze exonuclease ladders may be a particularly promising approach.1,2 Structure methods are particularly important in cancer and mutagenesis studies in which modifications of low picomole amounts of modified oligodeoxynucleotides must be determined. The most common approach to determine modified oligodeoxynucleotides and DNA is gel electrophoresis. The main limitation associated with the gel methods, however, is that the gel casting and electrophoresis are time-consuming.5 Moreover, the classical methods have limited applicability because mass accuracy is low. Modified oligodeoxynucleotides can also be determined by a 32P-postlabeling assay6 (but standards must be * To whom correspondence should be addressed. Email: mgross@ wuchem.wustl.edu. (1) Limbach, P. A. Mass Spectrom. Rev. 1997, 15, 297-336. (2) Nordhoff, E.; Kirpekar, F.; Roepstorff, P. Mass Spectrom. Rev. 1997, 15, 69-138. (3) Schuette, J. M.; Pieles, U.; Maleknia, S. D.; Srivatasa, G. S.; Cole, D. L.; Moser, H. E.; Afeyan, N. B. J. Pharm. Biomed. Anal. 1995, 13, 1195-1203. (4) Muddiman, D. C.; Smith, R. D. Rev. Anal. Chem. 1998, 17, 1-68. (5) Hahner, S.; Ludemann, H.-C.; Kirpekar, F.; Nordhoff, E.; Roepstorff, P.; Galla, H.-J.; Hillenkamp, F. Nucleic Acids Res. 1997, 25, 1957-1964. 10.1021/ac010042l CCC: $20.00 Published on Web 06/19/2001

© 2001 American Chemical Society

available to calibrate the method) and by atomic force microscopy.7 Although tandem mass spectrometry is also a means to sequence modified oligodeoxynucleotides, there are only a few examples, and most have been focused on small oligodeoxynucleotides (up to 12 bases). For example, Stemmler et al.8 applied MALDI-FTMS to the structural characterization of modified oligonucleotides 4-, 6-, and 11-mers. They showed that the complete base sequence and the location of a PAH-modified base could be determined for a tetramer and two hexamers. Extensive fragmentation that accompanied the desorption of 11-mers prevented their sequence determination.8 We recently reported that both ESI with MS/MS and MALDI combined with postsource decay (PSD) distinguish normal and UV photodamaged oligodeoxynucleotides containing 4-8 bases.9-11 One problem with direct sequencing by MS/MS is that fragmentation on many instruments is not complete for large oligodeoxynucleotides. A second is that fragmentation induced by collisional activation of ESI-produced ions or following the PSD of MALDI-produced ions of oligodeoxynucleotides depends on sequence, and this means that the sequence information may be incomplete or difficult to interpret for some sequences. These limitations prevent the application of MS/MS for sequencing large oligodeoxynucleotides (more than 12 bases), particularly when low picomole detection limits are required. Thus, there remains a need to develop alternative sequencing methods. The exonuclease ladder method provides such an opportunity for improvement. Pieles and co-workers12 successfully determined a 12-base oligonucleotide sequence (5′-[GCTTXCTCGAGT], X ) 2′-O-methyl adenosine) by MALDI and enzyme digestion. Keough et al.13 developed a method that includes nuclease digestion to characterize oligodeoxynucleotides containing modified phosphate groups along the backbone. Similar approaches have been further (6) Randerath, K.; Reddy, M. V.; Gupta, R. C. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 6126-6129. (7) Sun, H. B.; Qian, L.; Yokota, H. Anal. Chem. 2001, 73, 2229-2232. (8) Stemmler, E. A.; Buchanan, M. V.; Hurst, G. B.; Hettich, R. L. Anal. Chem. 1995, 67, 2924-2930. (9) Vollmer, D.; Zhao, X.; Taylor, J.-S.; Gross, M. L. Int. J. Mass Spectrom. Ion Processes 1997, 165, 487-496. (10) Wang, Z.; Wan, K. X.; Ramanathan, R.; Taylor, J. S.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1998, 9, 683-691. (11) Wang, Y.; Taylor, J.-S.; Gross, M. L. J. Am. Soc. Mass Spectrom. 1999, 10, 329-338. (12) Pieles, U.; Zurcher, W.; Schar, M.; Moser, H. E. Nucleic Acids Res. 1993, 21, 3191-3196.

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developed to analyze other modified oligodeoxynucleotides.3,14-20 Most recently, Cadet and co-workers21-24 also applied MALDItime-of-flight (TOF)MS combined with enzyme digestion and ESIMS to investigate the biological significance of oxidized lesions in DNA. An important class of modified oligonucleotides possess apurinic/apyrimidinic (AP) sites.25-29 AP sites are either generated spontaneously or produced by exposure of cells to certain damaging chemicals or radiation.26-29 The structure of synthetic oligodeoxynucleotides with AP sites shows that the backbone phosphodiester bonds on either side of an AP site are distorted.29 This distortion may cause misreading of nucleotide sequences, leading to toxicity and mutagenicity.27,30,31 One hypothesis for lesion formation in breast and lung cancers is that metabolic activation of a procarcinogen (i.e., an estrogen or polycyclic aromatic hydrocarbon, respectively) leads to damage to DNA, via AP-site formation;25,32 therefore, the goals of the research reported here are to sequence modified oligodeoxynucleotides, locate their AP sites, and measure the kinetics for the enzyme reactions by using a combined approach of MALDI and exonuclease ladders. Although optical spectroscopic methods are usually applied for enzyme kinetic studies, mass spectrometry is more sensitive and possibly more convenient. In early studies, Caprioli and coworkers33 reported that fast atom bombardment mass spectrometry can be used to follow trypsin digestion reactions in real-time inside the instrument. More recently, Dizdaroglu et al.34 investigated the kinetics of excision of purine lesions from DNA by using gas chromatography, isotope-dilution (GC/ID) MS. Simpson and co-workers35,36 reported that stopped-flow ESI-MS can be applied (13) Keough, T.; Shaffer, J. D.; Lacey, M. P.; Riley, T. A.; Marvin, W. B.; Scurria, M. A.; Hasselfield, J. A.; Hesselberth, E. P. Anal. Chem. 1996, 68, 34053412. (14) Tamura, T.; Araki, Y.; Yamaoka, S.; Inagaki, K.; Tanaka, H. Nucleic Acids Res. 1997, 25, 4162-4164. (15) Wu, H.; Chan, C.; Aboleneen, H. Anal. Biochem. 1998, 263, 129-138. (16) Wu, H.; Aboleneen, H. Anal. Biochem. 2000, 287, 126-135. (17) Wu, H.; Aboleneen, H. Anal. Biochem. 2001, 290, 347-352. (18) Zhang, L. K.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2000, 11, 854-865. (19) Asara, J. M.; Hess, J. S.; Lozada, E.; Dunbar, K. R.; Allison, J. J. Am. Chem. Soc. 2000, 122, 8-13. (20) Tretyakova, N. Y.; Niles, J. C.; Burney, S.; Wishnok, J. S.; Tannenbaum, S. R. Chem. Res. Toxicol. 1999, 12, 459-466. (21) Cadet, J.; Delatour, T.; Douki, T.; Gasparutto, D.; Pouget, E.-P.; Ravanat, J.-L.; Sauvaigo, S. Mutat. Res. 1999, 424, 9-21. (22) Gasparutto, D.; Bourdat, A.-G.; D’Ham, C.; Duarte, V.; Romieu, A.; Cadet, J. Biochimie 2000, 82, 19-24. (23) Romieu, A.; Gasparutto, D.; Molko, D.; Ravanat, J.-L.; Cadet, J. Eur. J. Org. Chem. 1984, 49, 9-56. (24) D’Ham, C.; Romieu, A.; Jaquinod, M.; Gasparutto, D.; Cadet, J. Biochemistry 1999, 38, 3335-3344. (25) Cavalieri, E. L.; Stack, D. E.; Devanesan, P. D.; Todorovic, R.; Dwivedy, I.; Higginbotham, S.; Johansson, S. L.; Patil, K. D.; Gross, M. L.; Gooden, J. K.; Ramanathan, R.; Cerney, R. L.; Rogan, E. G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10937-10942. (26) Loeb, L. A.; Preston, B. D.; Snow, E. T.; Schaaper, R. M. Basic Life Sci. 1986, 38, 341-347. (27) Loeb, L. A.; Preston, B. D. Annu. Rev. Genet. 1986, 20, 201-230. (28) Zhou, W.; Doetsch, P. W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 66016605. (29) Doetsch, P. W.; Cunningham, R. P. Mutat. Res. 1990, 236, 173-201. (30) Doetsch, P. W.; Viswanathan, A.; Zhou, W.; Liu, J. NATO ASI Ser., Ser. A 1999, 302, 97-110. (31) Zhou, W.; Doetsch, P. W. Ann. N.Y. Acad. Sci. 1994, 726, 351-354. (32) Nutter, L. M.; Ngo, E. O.; Abul-Hajj, Y. J. J. Biol. Chem. 1991, 266, 1638016386. (33) Smith, L. A.; Caprioli, R. M. Biomed. Mass Spectrom. 1983, 10, 98-102. (34) Karakaya, A.; Jaruga, P.; Bohr, V. A.; Grollman, A. P.; Dizdaroglu, M. Nucleic Acids Res. 1997, 25, 474-479.

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to study the kinetics of conversion of glucose to glucose-6phosphate catalyzed by hexokinase. Most relevant to this work is the report of Bentzley et al.,37 who applied MALDI-MS to measure the rate of disappearance of a series of 5- and 7-base oligodeoxynucleotides caused by calf spleen phosphodiesterase. They did not measure, however, the rates of appearance of any products, and that is a major point of the research reported here. The rates of hydrolysis of DNA and oligodeoxynucleotides by an exonuclease were determined previously by fluorescence spectroscopy and HPLC.38 The fluorescent base analogue, 2-aminopurine (2-AP), is one of the compounds most widely used as a probe in the kinetic measurement of nucleotide insertion and excision.38-40 Rates of nucleotide removal are measured by following the increase in the environmentally sensitive fluorescence for 2-AP.38 This method, however, cannot be directly applied to modified DNA or oligodeoxynucleotides unless the rates of removal of d(2-APMP) from the modified base are known. Another approach that is suitable for small oligodeoxynucleotides is HPLC. Maccubbin et al.41 investigated the effect of specific radiation-induced lesions on the digestion of a series of dinucleotide monophosphates by SVP or BSP. They used HPLC elution profiles to follow the rates of enzyme digestion of the dinucleoside monophosphates. Because the elution profile becomes very complex when the DNA strand becomes longer, an alternative approach is needed for the kinetic studies. MATERIALS AND METHODS Materials. Water was pretreated with a Milli-Q (Millipore, Bedford, MA) ultrapure water filtration system before its use as solvent. The anthranilic acid and nicotinic acid were obtained from Aldrich Chemical Co. (Milwaukee, WI). Both acids were purified by recrystallization from water. Decolorizing charcoal was used to remove impurities during the recrystallization. Cation-exchange beads in the NH4+ form were prepared from chromatography beads (AG50W-X8, 100-200 mesh; Bio-Ad, Melville, NY) in the H+ form according to the literature.42 The oligonucleotide, H21 (Table 1), and internal standards [d(T5), d(T10), d(T18), d(T20)] were synthesized at the Nucleic Acid Chemistry Laboratory, Washington University School of Medicine. The oligonucleotides, R7X and R6XX (Table 1), were provided by Professor J.-S. Taylor of the Department of Chemistry at Washington University. All of the other oligonucleotides in Table 1 were from Professor C. Hunt of the Mallinckrodt Institute of Radiology, Washington University. Snake venom phosphodiesterase (SVP, phosphodiesterase I) was purchased from Pharmacia Biotech (Piscataway, NJ), and bovine spleen phosphodiesterase (BSP, phosphodiesterase II) was obtained from Sigma Chemical Co. (St. Louis, MO). (35) Simpson, F. B.; Northrop, D. B. Mass Spectrom. Biol. Med. 2000, 329364. (36) Northrop, D. B.; Simpson, F. B. Bioorg. Med. Chem. 1997, 5, 641-644. (37) Bentzley, C. M.; Johnston, M. V.; Larsen, B. S. Anal. Biochem. 1998, 258, 31-37. (38) Linn, S. M.; Lloyd, R. S.; Roberts, R. J. Nucleases, 2nd ed.; Cold Spring Harbor Laboratory Press: New York, 1993; Chapter 8. (39) Nordlund, T. M.; Andersson, S.; Nilsson, L.; Rigler, R.; Graeslund, A.; McLaughlin, L. W. Biochemistry 1989, 28, 9095-9103. (40) Weinfeld, M.; Soderlind, K. J. M.; Buchko, G. W. Nucleic Acids Res. 1993, 21, 621-626. (41) Maccubbin, A.; Evans, M.; Paul, C. R.; Budzinski, E. E.; Przybyszewski, J.; Box, H. C. Radiat. Res. 1991, 126, 21-26. (42) Wang, B. H.; Biemann, K. Anal. Chem. 1994, 66, 1918-1924.

Table 1. Sequence of Oligodeoxynucleotides Submitted to the Ladder Methoda oligo name

sequence

H21 H7X H12X H13X H14X H15X R7X R6XX

5′-d(CGC TTG ATG AGT CAG CCG GAA) 5′-d(CGC TTG XTG AGT CAG CCG GAA) 5′-d(CGC TTG ATG AGX CAG CCG GAA) 5′-d(CGC TTG ATG AGT XAG CCG GAA) 5′-d(CGC TTG ATG AGT CXG CCG GAA) 5′-d(CGC TTG ATG AGT CAX CCG GAA) 5′-d(GAG TAT XAT GAG) 5′-d(GAG TAX XAT GAG)

a

X indicates the abasic site.

Instrumentation. MALDI-TOF spectra were acquired on a Voyager-DE RP (PerSeptive Biosystems; Framingham, MA) mass spectrometer equipped with a nitrogen laser at 337 nm. All of the spectra were obtained in the negative ion mode at an accelerating voltage of 25 kV. Oligodeoxynucleotide molecular weight determination required approximately 40 laser shots, whereas 100225 shots were summed when oligodeoxynucleotide digests were analyzed. MALDI Sample Preparation. The MALDI matrix was prepared by dissolving nicotinic acid, anthranilic acid, and ammonium citrate at molar ratios of 2:1:0.003 in acetonitrile:water (2:1).18 Samples were prepared by mixing 1 µL of analyte and 2 µL of matrix solution on the MALDI plate and air-drying. Solutions of 0.2 µL of 1 µM d(T5), d(T10), d(T18) or d(T20) were used to calibrate the m/z scale of the mass spectrometer. Enzymatic Digestion. A 1-µL aliquot of the sample containing 10 pmol of 12-mers or 40 pmol of the 21-mers (Table 1) was used in both SVP and BSP digestion experiments. For the SVP digestion, 1.0-2.0 µL of SVP solution (1 × 10-2 units/µL) was added to 1 µL of the oligodeoxynucleotide solution, 6 µL of 100 mM ammonium citrate (pH was adjusted to 9.4 with NH4OH), and 6 µL of H2O. For the BSP digestion, 1-2 µL BSP (1 × 10-2 units/µL) solution was added to 1 µL of the oligodeoxynucleotide solution and 7 µL of H2O. For the time-dependent studies, digest solution was maintained at 37 °C for the SVP digestion and kept at room temperature for the BSP digestion. A 0.5-µL aliquot of the digest solution was removed every 1 min during the first 10-12 min, and every 5-20 min thereafter over a total digestion period of 60 min. The sample was held on dry ice for 5 s to quench the digestion, then mixed with 1.0 µL of the matrix solution, and immediately spotted on the MALDI plate and air-dried for analysis. An extra 0.4 µL of the matrix solution was added to the dried sample spots if better sensitivity was needed. To evaluate the precision of the method, we applied 1.0 µL of SVP solution (1 × 10-2 units/µL) to digest 40 pmol of the H21 (Table 1) at 37 °C. A 0.5-µL aliquot of the digest solution was removed every 1 min during the first 10 min, and every 2 min thereafter over a total digestion period of 16 min. The MALDI sample spots were prepared as described above. Each mass spectrum was generated from data accumulated from 60 laser shots, and a total of 13 spectra were recorded for the kinetic studies. The experiment was repeated two more times, and the three sets of spectra were collected and submitted for mathematic treatment, as described below.

Analysis of the Kinetic Data. Three basic steps were used to process the time-dependent MALDI mass spectra. The first was to normalize the intensities at each time point so that their sum was equal to 1. The second was to execute a loop to minimize the error between the time trajectory of a postulated model system and the normalized intensities. The third step was to integrate the system with solution rate constants to obtain intensities as a function of time for checking the reasonableness of the fit. The model system describes the oligodeoxynucleotide concentrations from a sequence of enzyme-catalyzed consecutive reactions. The concentrations are represented as the coordinates in a state vector y as shown in eq 1. The first coordinate, I1, gives the concentration of the undigested oligodeoxynucleotide. For the purposes of the model, the reaction was assumed to start at time zero, in which case the concentrations are given by y(0) in eq 2. The concentration of the starting material was chosen to be 1 to correspond to the normalized experimental intensities.

[]

I1 I2 ‚ y) ‚ ‚ In

(1)

[]

1 0 ‚ y(0) ) ‚ ‚ ‚ 0

(2)

Each reaction in the chain was assumed to be first-order and described by a rate constant. The rate constant of the first reaction was given by k1, and the rate constants of the subsequent reactions were denoted with increasing subscripts. The simple first-order differential equations that describe each reaction step can be conveniently collected together into a state equation, which gives the time rate of change for the concentrations as shown in eq 3 as a function of the concentrations. Equation 3 is in a form suitable for numerical integration by standard methods.43 Given a postulated set of rate constants and the above initial condition, a trial time trajectory of concentrations was computed. The integration was performed with the “Rkadapt” in Mathcad 8 Professional44 (MathSoft, Inc., Cambridge, MA) with 20 steps per minute for a time sufficient to include the experimental times.

[ ]

-k1I1 k1I1 - k2I2 ‚ dy ) ‚ dt ‚ kn-1In-1 - knIn

(3)

The rate constants for a particular experiment were solved by a search that minimizes the error between calculated and experimental values. For each trial in the search, the error was computed as the sum of the squares of the differences (residuals) at the (43) Press, W. H.; Teukolsky, S. A.; Vetterling, W. T.; Flannery, B. P. Numerical Recipes in C, The Art of Scientific Computing, 2nd ed.; Syndicate of the University of Cambridge: Cambridge, 1992. (44) MathSoft, Mathcad User’s Guide, Mathcad 8 Professional; MathSoft, Inc.: Cambridge, MA, 1998.

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Figure 1. Time-dependent MALDI mass spectra of the SVP digestion products of 5′-d(CGC TTG ATG AGT CAG CCG GAA) (H21).

Chart 1

experimental times between the numerically integrated concentrations and the normalized spectra intensities. If the experimental intensity for a concentration at an experimental time point was zero, no contribution was made to the calculated error. The search was implemented with the “Minimize” function in Mathcad, which postulates the rate constants for each trial of the search. The rate constants were constrained to be nonnegative. All of the algebraic details of the above process were handled in the Mathcad spreadsheet. RESULTS AND DISCUSSION Molecular Weight and Stability Determination. To develop a MALDI-TOF method to determine oligodeoxynucleotides that 3266

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possess an abasic site, we build on the use of MALDI to determine chemically modified oligodeoxynucleotides. Further, we designed a set of appropriately modified oligodeoxynucleotides (Table 1) on the basis of the nuclear transcription factor activator protein 1 (AP-1), which plays an important role in human genetic studies. In the past 10 years, more than 1000 papers focusing on the AP-1 DNA binding activity were published; therefore, we included the oligodeoxynucleotides H7X to H15X (Table 1), which constitute a series of synthetic 21-mers. These modified oligodeoxynucleotides have a sequence that is based on the AP-1 DNA core binding region, TGAGTC, but contain abasic sites located at different positions. We employed the dSpacer (Chart 1) to introduce model abasic sites into these oligodeoxynucleotides, because previous studies45-47 showed that dSpacer mimics abasic sites in oligodeoxynucleotides. Further, an oligodeoxynucleotide containing such an abasic site is more stable and easier to work with than one containing an abasic site in which the nucleobase is replaced with an OH group. The first step was to verify the molecular weight of the modified oligodeoxynucleotides and to test their stability. We recently reported that the combination of anthranilic acid and (45) Takeshita, M.; Eisenberg, W. Nucleic Acids Res. 1994, 22, 1897-1902. (46) Takeshita, M.; Chang, C. N.; Johnson, F.; Will, S.; Grollman, A. P. J. Biol. Chem. 1987, 262, 10171-10179. (47) Kalnik, M. W.; Chang, C. N.; Grollman, A. P.; Patel, D. J. Biochemistry 1988, 27, 924-931.

Figure 2. Time-dependent MALDI mass spectra of the SVP digestion products of 5′-d(CGC TTG XTG AGT CAG CCG GAA) (H7X).

nicotinic acid with diammonium citrate (2:1:0.003) offers improvement for the MALDI analysis of oligonucleotides at low concentration.18 Applying the same matrixes, we confirmed the molecular weight of all of the modified oligodeoxynucleotides listed in Table 1 (spectra not shown). The mass we found for these oligos was within 1 Da when a resolving power of approximately 800 (full width at half-maximum) could be achieved. Neither fragments nor impurities could be detected throughout the low-mass range of the spectra, and this finding assured us that the samples were chemically pure and suitable for further experiments. To test sample stability, we held these model oligonucleotides at room temperature for a 4-h period and repeated the MALDI analysis. We did not detect any increase in the amount of the fragment ions. This indicated that the spontaneous hydrolysis of these modified oligodeoxynucleotides with AP sites would not interfere with the enzymatic digestion experiments that we employed to locate the abasic site. Sequence and Location of the AP Sites in Oligodeoxynucleotides. An early example of structure determination of a dinucleotide containing an abasic site is by Paterson and coworkers,48 who treated two isomers, 5′-d(XA) and 5′-d(AX) with

SVP and examined by HPLC the response of the nuclease to the abasic site. We chose to evaluate an alternative sequencing procedure using MALDI and exonuclease digestion, because we wished to work with oligomers that have as many as 21 nucleotides. In a test of the efficacy of enzymatic digestion, we submitted unmodified oligodeoxynucleotides H21 (Table 1) to partial enzymatic digestion using two enzymes, SVP and BSP, that remove one nucleotide at a time from the 3′ and 5′ end, respectively. The time-dependent MALDI-TOF mass spectra of SVP digestion products of H21 (Figure 1) show that the first six nucleotides were hydrolyzed in 1 min. After 5 min, nucleotides at positions 5-13 were released, and the sequence information from base position 1 to position 18 was available within 14 min. We also submitted the H21 to BSP digestion (spectra not shown), and within 5 min, the sequence of 19 nucleotides was available from the 5′ end. These results are consistent with those we reported previously on the SVP and BSP digestion of unmodified oligodeoxynucleotides.18 The accuracy of the mass differences between adjoining (48) Weinfeld, M.; Liuzzi, M.; Paterson, M. Nucleic Acids Res. 1989, 17, 37353745.

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Figure 3. Time-dependent MALDI mass spectra of the BSP digestion products of 5′-d(CGC TTG XTG AGT CAG CCG GAA) (H7X).

peaks in the spectra (Figure 1) was ( 0.5 Da, allowing an unambiguous and facile determination of each base residue. To locate the position of the AP site, we submitted a modified 21-mer, H7X (Table 1), to SVP digestion under the same reaction conditions as described for hydrolysis of H21. The time-dependent mass spectra (Figure 2) show that the SVP digestion of H7X was slower than that of H21. For example, 4 min was required to release the first six nucleotides for H7X, whereas only 1 min was required for H21. Moreover, the digestion could not pass the model AP site even after we treated the H7X to SVP for as long as 50 min (Figure 2). The SVP digestion of H15X (Table 1), which has the AP site at a different location, stopped after producing the product, 5′-d(CGCTTGATGAGTCAX). The other oligodeoxynucleotides in Table 1 gave similar results (spectra not shown). The SVP digestion rate clearly slows down as the digestion approaches the AP site, and this reduction in rate indicates that the modification site is nearby. We also evaluated the BSP digestion of the oligodeoxynucleotides in Table 1 to demonstrate that use of this enzyme also identifies model AP sites. After a 3-min digestion of H7X with BSP (Figure 3), peaks ranging from 1 to 7 appeared. Compared to the BSP digestion of the unmodified H21, the digestion of H7X is clearly inhibited by the AP site (note peak 7 in all of the spectra). 3268

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The digestion rate becomes too small to measure for cleavage of the phosphodiester bond that adjoins the AP site, even after extended treatment (40 min) of the H7X with the BSP. The rate of BSP digestion of other modified oligodeoxynucleotides in Table 1 also slowed near the AP sites (spectra not shown). Such inhibition coupled with mass spectral information using the complementary enzyme permits unambiguous location of the AP sites in the strands. We expected that applying an excess of the enzyme might increase the digestion rates of the modified oligodeoxynucleotides, causing some confusion in locating the AP site. To test the effect of increased enzyme, we doubled its concentration (to 3 × 10-2 units/40 pmol oligodeoxynucleotides) in the SVP digestion of H12X (Table 1). We found that the first nine nucleotides of H12X were released within 10 min and that the rate slowed, as expected, at the AP site (spectra not shown). We then followed the same digestion by MALDI MS for an additional 50 min (Figure 4) and found, to our surprise, that the SVP acted as an endonuclease to catalyze hydrolysis of the digestion product of H12X, 5′-d(CGCTTGATGAGX) (1′)(Figure 4). Peaks corresponding to 5′-d(CGCTTGATGAGX) after cleavage of C (2′), then G (3′), C (4′), T (5′), T (6′), G (7′), and A (8′) were observed. After 45 min, 5′d(TGATGAGX) became the main product, and the differences in

Figure 5. Time-dependent MALDI mass spectra of digestion products of 5′-d(GAG TAT XAT GAG) (R7X) produced by using excess (1.5 µL) BSP (1.0 × 10-2 units/µL). Figure 4. Time-dependent MALDI mass spectra of the digestion products of 5′-d(CGC TTG ATG AGX CAG CCG GAA) (H12X) produced by using excess (3 µL) SVP (1 × 10-2 unit/µL). The use of primes for peak numbers indicates that the peaks represent endonuclease digestion products.

the relative abundances of the products may reflect the influences of secondary structure on the digestion rate. The digestion products of H13X and H14X (Table 1) also began to hydrolyze after a short delay when submitted to the same treatment (spectra not shown). The resumption of an endonuclease activity requires an excess of SVP and longer digestion time. This endonuclease activity was previously reported by Yum49,50 and Pritchard.51 More recently, Cadet and co-workers52 also described similar results when they incubated an 8-oxo-7,8-dihydro-2′-deoxyguanosine modified oligodeoxynucleotide with SVP for over 60 min. Nevertheless, we cannot rule out some contamination of other enzymes that have endonuclease activity. The digestion of the modified oligodeoxynucleotides with excess BSP gave different results as compared to those from the use of SVP. The BSP (1.5 × 10-2 units/40 pmol oligos) digestion of R7X (Table 1) progressed to the AP site (6) within 1 min and passed it, although inhibited, within 3 min. The mass difference between peaks 6 and 7 (Figure 5), 180.6, agrees well with the calculated value for a sugar-phosphate backbone (180.2). The peak labeled “-GH” represents a fragment of R7X in which the (49) Yum, Y. N.; Kim, D. S. Han’guk Saenghwa Hakhoechi 1993, 26, 252-257. (50) Yum, Y. N.; Kim, D. S. Han’guk Saenghwa Hakhoechi 1993, 26, 258-263. (51) Pritchard, A. E.; Kowalski, D.; Laskowski, M. J. Biol. Chem. 1977, 252, 8652-8659. (52) Bourdat, A.-G.; Gasparutto, D.; Cadet, J. Nucleic Acids Res. 1999, 27, 10151024.

5′-guanine base is lost. The small peaks marked with stars in the spectra correspond to the potassium and the sodium adducts of the oligonucleotides. These adducts are also seen for the other lower abundance fragments produced in the digestion, although they are not labeled in the spectra. For R6XX, which has the same strand of R7X but contains an XX segment (X ) AP site), both the SVP and BSP digestion, even with excess enzyme, stopped at the site modification. Figure 7 shows the MALDI spectra of SVP (A) and BSP (B) digestion products of 5′-d(GAG TAX XAT GAG). We also applied the MALDI ladder method to an olignucleotide T6X′ (5′-TTT TTX′ TTT TTT) containing an abasic site in which an OH replaces the nucleobase (these abasic sites are the ones produced in vivo). To locate the position of the AP site, we submitted the T6X′ (of m/z 3479) to SVP digestion under the same conditions as described for H21. After 30 min, the reaction stopped, as indicated by the production of the m/z 1654 ion (5TTT TTX′), clearly revealing the modification site. We used 6-aza2-thiothymine instead of AA/NA as the MALDI matrix here, because the anthranilic acid can react with an abasic site containing an OH instead of the base. Using HPLC, Paterson and co-workers48 found that SVP readily cleaved 5′-d(X′A) to 5′-dAMP but had no discernible effect on 5′d(AX′). Our results are consistent with their observation. Moreover, the ability to detect AP sites in larger oligodeoxynucleotides with high sensitivity suggests that mass spectrometry is a better choice than HPLC for a routine AP-site screen. Kinetic Analysis of the Results of SVP and BSP Digestion. The relative ease of obtaining time-dependent spectra points to the use of MALDI MS for following the kinetics of enzymeAnalytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Figure 6. MALDI mass spectra of the digestion products of 5′-d(GAG TAX XAT GAG) (R6XX): (A) SVP (5 × 10-2 units) and (B) BSP (1 × 10-2 units).

catalyzed reactions. In this section, we describe the development of a mathematical treatment of the time-dependent digestion results and an evaluation of results found in establishing the precision of the outcome. The data generated by a MALDI ladder method may be the basis for a general and rapid means for obtaining rate constants for complicated, enzyme-catalyzed consecutive reactions. An advantage of using MALDI instead of ESI mass spectrometry to investigate the enzymatic digestion of oligodeoxynucleotides is the simplicity of the MALDI spectrum. Moreover, gas-phase enzyme-substrate complexation may occur upon ESI, obfuscating what happens in solution. MALDI, on the other hand, allows for a fast quench of the enzyme digestion followed by a simple workup and analysis to give uncomplicated mass spectra. Enzymatic processes are multistep transformations involving, at the minimum, substrate-enzyme binding, substrate-enzyme to substrate-product transformation, and substrate-product dissociation (eq 4).53,54 Transition-state theory indicates that the interconversion of the enzyme-substrate complex to the enzymeproduct complex is the rate-limiting step.54,55 This leads to the

well-known Michaelis-Menten relationship (eq 5). The ratio Km (eq 6) is known as the

(53) Steinfeld, J. I.; Francisco, J. S.; Hase, W. L. Chemical Kinetics and Dynamics; Prentice Hall: Englewood Cliffs, NJ; 1995; Chapter 5. (54) Mader, M. M.; Bartlett, P. A. Chem. Rev. 1997, 97, 1281-1301.

(55) Albery, W. J.; Knowles, J. R. Angew. Chem. 1977, 89, 295-304. (56) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Company: New York, 1996; Chapter 8.

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k1

kcat

E + S y\ z E‚S 98 E‚P f E + P k -1

V)

kcat [E][S] [S] + Km

Km )

k-1 + kcat k1

for [S] , Km, V )

kcat [E][S] Km

V ) k[S]

(4) (5) (6) (7) (8)

Michaelis Constant. At very low substrate concentration, when [S] is much less than Km, the Michaelis-Menten equation can be reduced to eqs 7 and 8; that is, the rate is directly proportional to the substrate concentration.56 Thus, we can treat the SVP or BSP

digestion of oligodeoxynucleotides as consecutive pseudo-firstorder processes. The kinetic method we wish to use is based on our recent demonstration that MALDI can be used for quantitative analysis of small- to middle-size (8- to 18-mer) oligodeoxynucleotides at the low-picomole to femtomole level.18 Plots of the amount of analyte against the analyte/internal standard peak-height ratio gave a linear calibration curve at least over a concentration range of 0.2 to 5 µM in the original solution. This ensures us that we can monitor accurately the kinetics of enzymatic degradation over this range. Although a mixture of oligodeoxynucleotides is observed at any given time point, only peaks over a narrow mass range have nonzero intensities, and we expect their sensitivity factors to be nearly identical. Therefore, uniform response over a wide mass range is not a requirement, and we suggest that calibration standards are not necessary. An important requirement for kinetic measurements by a batch method is to terminate effectively the enzyme digestion at each time point. We quenched the digestion by decreasing abruptly the temperature and evaporating the solvent immediately after the sample was spotted on the MALDI plate. We then tested the effectiveness of the quench by evaporating the solvent and allowing the samples to sit for 12 h inside the MALDI mass spectrometer. A new mass spectrum was found to be nearly identical to the original. The kinetic parameters of the enzymatic digestion were established by computer curve-fitting,57-63 using the consecutive reaction model. Others developed various computer programs for least-squares curve-fitting and applied them to the analysis of enzyme kinetic data.64-66 For example, Frieden and co-workers67-69 have developed two kinetic simulation and fitting programs, namely KINSIM and FITSIM, that can be downloaded from their website (http://biochem.wustl.edu/∼cflab). A description of the model we used to recover the rate constants is in the Experimental Section. The model we use is similar in principle to those used in the past. The reason we chose Mathcad to implement a solution is that it presents to the user the algorithm in standard mathematical notation. Moreover, the user can easily make changes to the algorithm if so desired. Kinetics of Hydrolysis of Unmodified Oligodeoxynucleotides. We applied the method to both the SVP and BSP digestion of the unmodified H21 (Table 1). The fitted curves clearly show the kinetics of hydrolysis of the test oligonucleotide, H21 (spectra not shown). The 21-mer concentration decreases with increasing digestion time, and the concentrations of the products of digestion increase, then decrease as they are digested, to give smaller oligos. We found that the calculated rate curves include most of the (57) Kahley, M. J.; Novak, M. J. Chem. Educ. 1996, 73, 359-364. (58) Reich, L. Thermochim. Acta 1996, 273, 113-118. (59) Teraoka, R.; Otsuka, M.; Matsuda, Y. Pharm. Res. 1994, 11, 1077-1081. (60) Aboul-Seoud, A.; Amer, A. Chem. Eng. World 1993, 28, 199-201. (61) Chrastil, J. Comput. Chem. 1993, 17, 103-106. (62) Chrastil, J. Comput. Chem. 1988, 12, 289-292. (63) Tadi, M.; Yetter, R. A. Int. J. Chem. Kinet. 1998, 30, 151-159. (64) Mottola, H. A.; Perez-Bendito, D. Anal. Chem. 1996, 68, 257-289. (65) Crouch, S. R.; Scheeline, A.; Kirkor, E. S. Anal. Chem. 2000, 72, 53-70. (66) Crouch, S. R.; Cullen, T. F.; Scheeline, A.; Kirkor, E. S. Anal. Chem. 1998, 70, 53R-106R. (67) Dang, Q.; Frieden, C. Trends Biochem. Sci. 1997, 22, 317. (68) Zimmerle, C. T.; Frieden, C. Biochem. J. 1989, 258, 381-387. (69) Barshop, B. A.; Wrenn, R. F.; Frieden, C. Anal. Biochem. 1983, 130, 134145.

Table 2. Pseudo-First-Order Rate Constants Calculated by the Kinetic Treatment of Time-Dependent MALDI Mass Spectra H21 (SVP)a

H14X (SVP)a

H21 (BSP)a

H14X (BSP)a

rate const

kb min-1

base

kb min-1

base

kb min-1

base

kb min-1

k0 k1 k2 k3 k4 k5 k6 k7 k8 k9 k10 k11 k12 k13 k14 k15 k16 k17

1.4 2.0 2.8 2.5 0.82 0.85 1.3 1.2 0.9 1.3 1.1 1.3 1.0 1.3 1.3 0.73 0.66 1.3

A A G G C C G A C T G A G T A G T T

A A G G C C G X C T G A G T A G T T

1.7 6.0 5.0 5.5 5.6 5.2 2.9 2.5 2.2 1.6 1.8 1.0 1.5 1.7 1.4 0.70 0.73 0.62

C G C T T G A T G A G T C A G C C G

1.2 2.1 2.3 1.8 1.3 1.6 1.0 c

0.72 0.87 0.70 0.75 0.58 0.57 0.38 0.33 0.29 0.23 0.33 0.34 0.17 c

base C G C T T G A T G A G T C X G C C G

a See Table 1 for name of the oligodeoxynucleotide. b Precision is (6%. c The rate becomes undetectably slow.

Figure 7. Fitted rate curve for the SVP digestion of H21 represented by the m/z 4898 ion.

experimental points (data not shown to relieve congestion), thus demonstrating the proposal that the rates are pseudo-first-order. The outcome of the kinetic method for H21 is two series of rate constants for cleavage at each nucleotide by SVP and BSP, respectively (Table 2). To examine the reproducibility of the MALDI analysis and the accuracy of the mathematical treatment, we repeated the SVP digestion of H21 (Table 1) three times on the same day. We then submitted the three sets of time-dependent MALDI data to Mathcad, treated them as before, and obtained three sets of rate constants. The statistics show that the average relative standard deviation for the rate constants is 6 ( 2%, which establishes the reproducibility of the method described here. A typical outcome (Figure 7) showing the experimental points and the calculated curve for the time dependence of the oligodeoxynucleotide represented by the m/z 4898 ion shows a good fit (Figure 7). The sums of the squares of the differences at the experimental times Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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between the numerically integrated concentrations and the normalized spectral intensities are 0.10, 0.12 and 0.12, respectively, for the three experimental trials. These errors were termed the “residual sum of the squares” (RSS) by others.70,71 To make more clear the goodness of the fit, we computed the root-mean-square (RMS) of the residuals as the measure of deviation between all of the experimental points (where an ion is detectable) and the calculated curve; the RMS is 0.033 ( 0.001 for all of the points. This small deviation shows that the kinetic model is appropriate for the rate data that we generated. We noticed that the reactivity of both enzymes appears to be specific for the nucleobase at the cleavage site. For example, the rate constant for hydrolysis of the G (k2 ) 2.8 min-1) at the 19th position from the 5′-end is similar to that of the G (k3 ) 2.5 min-1) at the 18th position but is 1.4 times greater than that of the A (k1 ) 2.0 min-1) at the 20th. The rate constant for hydrolysis of the G located deeper within the oligodeoxynucleotide (e.g., at position 11), however, drops to 1.1 min-1, giving evidence for the site specificity. Similar results were also obtained for the BSP digestion. The overall base specificity of the SVP or BSP digestion rate is summarized as G, T > A > C. This is consistent with reports from earlier studies on the determination of the rate of removing the first base from short oligodeoxynucleotides.37,72,73 Moreover, the initial reaction rates calculated from the rate constant generated in our experiments are also consistent with published data.37 To our knowledge, however, no kinetic studies of either SVP or BSP-catalyzed consecutive hydrolysis of DNA have been published to date. Comparison of the rate constants for SVP (pH ) 9.4, 37 °C) and BSP digestion (no buffer, room temp.) indicates that the BSP causes the first six bases of the oligodeoxynucleotide to hydrolyze more rapidly than does SVP, even at lower temperature (Table 2). This suggests that it is necessary to collect more data during the first 5 min to produce an accurate kinetic treatment of the BSP digestion ladder. Kinetics of Hydrolysis of Modified Oligodeoxynucleotides. Successful mathematical curve-fitting can be done for a 21-mer with an abasic site (H14X). One can clearly see that digestion ceases with the production of the m/z 4145 ion (a 14-mer) or the m/z 2310 ion (an 8-mer) in the BSP or SVP digestion, respectively, of the H14X, (Figure 8A,B). The sites of modification can be read directly from the fitted rate curves for either the BSP or the SVP digestion of H14X (Figure 8A,B). Generally, the rate constants for the digestion of the modified oligodeoxynucleotide (H14X) are smaller than those of the unmodified H21 (Table 2). This is even more obvious for the BSP digestion. The digestion rate constants for hydrolysis of H14X, which contains the abasic site, are on average 8 ( 1.5 times smaller than those of H21. An abasic site in an oligodeoxynucleotide has significant influence on the overall reactivity when the enzymes are acting on single-stranded DNA. The reduction in the reaction rate constants for SVP or BSP digestion of modified (70) Hocking, R. R. Methods and Applications of Linear Models: Regression and the Analysis of Variance; John Wiley & Sons: New York, 1996; 68-69. (71) Gutheil, W. G.; Kettner, C. A.; Bachovchin, W. W. Anal. Biochem. 1994, 223, 13-20. (72) Bernardi, A.; Bernardi, G. Centre Rech. Macromol, S. F. Biochim. Biophys. Acta 1968, 155, 371-377. (73) Bernardi, A.; Bernardi, G. Centre Rech. Macromol, S. F. Biochim. Biophys. Acta 1968, 155, 360-370.

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Figure 8. Fitted rate curves for: (A) the BSP digestion of H7X; (B) the SVP digestion of H7X.

oligodeoxynucleotides compared to that of normal oligodeoxynucleotides allows us to determine immediately that we are dealing with a modified oligonucleotide. CONCLUSIONS MALDI mass spectrometry is an appropriate method for determining rates and mechanisms of enzyme reactions. This may promote the investigation of point mutations in genomic DNA by comparing the kinetic properties of normal and modified DNA. The data (rate constants) may also yield insights into the biochemical basis for mutational hot and cold spots. The MALDIMS method can also be applied to study the biochemical properties of enzymes. In an earlier study, Weinfeld and coworkers40 used HPLC to examine the influence of nucleobase aromaticity on the kinetics of enzyme-substrate interactions. The MS method is a fast alternative to HPLC. A problem may arise because some enzyme-catalyzed reactions require high ionic strength or high concentration of specific metal ions. A sample cleanup must then be introduced to remove these components before the MALDI analysis. Many simple and rapid methods2,4 are available for the removal of these buffers and salts, including applying the commercially available ZipTips.74 The MALDI approach is simple and convenient for determining the kinetics of enzyme-catalyzed consecutive reactions of both modified and unmodified oligodeoxynucleotides. Kinetic data, such as those reported here, do lead to a better understanding of enzyme mechanism. Moreover, they provide guidance in the (74) Pluskal, M. G. Usa Nat. Biotechnol. 2000, 18, 104-105.

design of the MS ladder method for the detection of modified oligodeoxynucleotides in other biological studies. The MS ladder approach reported here may be applicable to the structure proof of oligodeoxynucleotides with other modifications (e.g., radiationinduced or chemically induced), and we are continuing the method development to determine oligodeoxynucleotides that have been modified photochemically or by steroid-quinone electrophiles. The method is limited by the requirements that the oligodeoxynucleotides are pure and available in 40 pmol quantities, although improvements in detection limits are expected. Application to larger oligonucleotides remains to be developed.

ACKNOWLEDGMENT The National Institutes of Health (Grant P01CA49210) and the National Centers for Research Resources of the National Institutes of Health (Grant P41RR00954) supported this research. We thank Professor Clayton Hunt for introducing us to the oligonucleotides containing a model abasic site and for providing samples. Received for review January 12, 2001. Accepted May 2, 2001. AC010042L

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