Anal. Chem. 1997, 69, 1107-1112
Chemical Sequencing of Phosphorothioate Oligonucleotides Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Lenore M. Polo, Tracy Donovan McCarley,† and Patrick A. Limbach*
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Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803
A new technique for sequencing phosphorothioate oligonucleotides is demonstrated that uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Current sequencing protocols for phosphorothioate analysis have drawbacks to their widespread implementation. Enzymatic and Maxam-Gilbert sequencing techniques require oxidation to the phosphodiester moiety, thereby increasing analysis time and eliminating the ability to locate modified linkages. Tandem mass spectrometry, which is finding increased use in phosphodiester oligonucleotide sequencing, has not been demonstrated on molecules with phosphorothioatelinked backbones. In the approach presented here, sequencing of the phosphorothioate-linked oligonucleotide is carried out using 2-iodoethanol to cleave at thiolated sites along the backbone. The technique has several advantages over current phosphorothioate sequencing methods: (1) sequencing is performed on the sample without prior oxidation; (2) both 5′ f 3′ and 3′ f 5′ mass ladders are generated, permitting bidirectional sequencing; (3) it is possible to determine the location of phosphorothioate linkages in mixed phosphorothioate/ phosphodiester oligonucleotides; (4) analysis times are short (e90 s); and (5) small sample amounts are used (2 h), the sample must be purified by size exclusion chromatography after the desulfurization step, and the method cannot distinguish between phosphorothioate and phosphodiester linkages. Another method for characterizing the sequence of synthetic oligonucleotides is the detection of failure sequences from the original synthesis step.8 This procedure takes advantage of the fact that automated solid phase synthesis of oligonucleotides is not always 100% efficient. Sequence determination of oligonucleotides by analysis of the failure sequences is an extremely simple and straightforward method. The mass spectrum will contain a series of peaks corresponding to the final product and each of the failure sequences, each of which differs in mass by the appropriate nucleotide residue value. While this method is relatively simple and straightforward, it is not applicable to cases in which a highly efficient synthesis is achieved with stepwise yields of 100% for the majority of the steps in the synthesis. In MS/MS, oligonucleotides are fragmented via collisionally induced dissociation to generate a number of product ions corresponding to the original parent ion.12 Sequence determination, particularly of large oligonucleotides, is a formidable problem in this technique because of the extensive number of fragmentation patterns that can occur from an oligonucleotide. Only recently have McCloskey and co-workers shown that this technique can be applied to an oligonucleotide of unknown sequence by using iterative steps that assign each nucleotide in the sequence on the basis of charge state and abundance rules.13 However, this method has not yet been shown to be effective in determining the sequence of backbone-modified oligonucleotides (including phosphorothioates). As noted by McCloskey and co-workers, this algorithm is intended for the sequence characterization of oligonucleotides at the 15-mer level and below. This size limitation is due to the limited fragmentation efficiencies of oligonucleotides larger than 15 residues in a triple-quadrupole mass spectrometer. Further, this method has only been demonstrated with an electrospray ionization source and a triple-quadrupole mass spectrometer. There remains some question as to the applicability of this sequencing algorithm for ions generated via MALDI and/ or for oligonucleotides analyzed using other types of mass analyzers. The results from McCloskey and co-workers are impressive and do demonstrate the inherent advantages of an MS/MS approach: speed of analysis, the ability to determine base or sugar modifications, and the ability to analyze mixtures of oligonucleotides. However, an enzymatic, chemical, or failure sequence approach also offers several important advantages for sequence determination: the ability to generate sequence information independent of the ionization source or mass analyzer used, speed of analysis (as will be demonstrated from the results here), and
ease of sequence interpretation due to the presence of a readily identified mass ladder. The sequencing approach developed here takes advantage of the susceptibility of the phosphorothioate backbone to alkylating agents.14,15 The reaction scheme is shown in Figure 1. There are three possible reaction pathways. Reaction path a results in a rearrangement to the phosphodiester moiety and is not useful for sequencing purposes. This pathway can be avoided by modifying the reaction time and concentration of alkylating agent used. Reaction paths b and c correspond to 5′- and 3′-sequence products, respectively. Thus, this chemical sequencing approach should generate a series of oligonucleotides differing in mass by the excised residue(s) (i.e., a mass ladder of peaks similar to that obtained using enzyme degradation or failure sequence analysis). This mass ladder should allow for ease of sequence interpretation. Further, this sequencing protocol is readily adaptable to different ionization sources and mass analyzers. Here we demonstrate the experimental protocol necessary to generate a mass ladder of ions from which the sequence of phosphorothioates can be determined.
(11) Schuette, J. M.; Pieles, U.; Maleknia, S. D.; Srivatsa, G. S.; Cole, D. L.; Moser, H. E.; Afeyan, N. B. J. Pharm. Biomed. Anal. 1995, 13, 1195-1203. (12) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70. (13) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal. Chem. 1996, 68, 1989-1999.
(14) Gish, G.; Eckstein, F. Science 1988, 240, 1520-1522. (15) Nakamaye, K. L.; Gish, G.; Eckstein, F.; Vosberg, H.-P. Nucleic Acids Res. 1988, 16, 9947-9959. (16) Xu, Q.; Musier-Forsyth, K.; Hammer, R. P.; Barany, G. Nucleic Acids Res. 1996, 24, 1602-1607. (17) Stec, W. J.; Zon, G.; Uznanski, B. J. Chromatogr. A 1985, 326, 263-280.
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EXPERIMENTAL SECTION Oligonucleotide Synthesis. Reagents for oligonucleotide synthesis were obtained from Glenn Research (Sterling, VA) and ChemGenes (Waltham, MA). Trityl-on oligonucleotides were synthesized using standard phosphoramidite chemistry on 1-µmol columns using a Perkin Elmer/Applied Biosystems (Foster City, CA) Model 394 DNA/RNA synthesizer. Introduction of the phosphorothioate linkage was carried out using 3-ethoxy-1,2,4dithiazolin-5-one (EDITH) at a concentration of 0.05 M.16 For each coupling, EDITH was delivered to the solid support column in two pulses in which the reactant was flushed through the column for 8 s and then allowed to react for 30 s. Average stepwise yields were 97% or better and were 100% for the majority of the synthesis steps of the mixed-base phosphorothioate oligonucleotides. OPC Purification. After synthesis, the deprotected oligonucleotides were purified using oligonucleotide purification cartridges (OPCs) purchased from Perkin Elmer to isolate the desired sequence from failure sequences. Purification is accomplished by washing the oligonucleotide and retaining the terminal dimethoxytrityl (DMT) protecting group. The DMTprotected oligonucleotide is retained on the OPC in 10% aqueous ammonium hydroxide. The OPC was washed with ammonium hydroxide to remove any impurities and failure sequences. The DMT group was then removed using 3% trifluoroacetic acid (TFA), and the remaining oligonucleotide was eluted with 20% acetonitrile. The eluate was evaporated to dryness on a LabConco centrivap (Kansas City, MO) and then reconstituted in 100 µL of nanopure water prior to analysis. HPLC Purification. If the OPC purification did not isolate the desired reaction product, the sample was further purified using reversed-phase high-performance liquid chromatography (HPLC). The buffers used for the separation were 0.1 M triethylammonium acetate, pH (buffer A), and acetonitrile (buffer B). The gradient was linear from 0% B to 100% B at 1%/min.17 HPLC fractions were evaporated to dryness on a centrivap and then reconstituted in 20 µL of Nanopure water prior to analysis.
Figure 1. Cleavage scheme proposed for the reaction of phosphorothioates with 2-iodoethanol.
Mass Spectrometry. The mass spectrometer used for analysis of the homologous phosphorothioate oligonucleotide and the mixed phosphodiester/phosphorothioate oligonucleotide was a PerSeptive Biosystems Inc. (Framingham, MA) Voyager linear MALDI-TOF instrument with an N2 laser. For the mixed-base phosphorothioate oligonucleotide, a PerSeptive Biosystems Inc. Voyager delayed extraction linear MALDI-TOF instrument with an N2 laser was used. A 2-µL aliquot of the oligonucleotide solution (∼10 mM) was added to 15 µL of a matrix solution consisting of a 2:1 ratio of 0.1 M diammonium hydrogen citrate and 0.5 M 2,4,6-trihydroxyacetophenone. Approximately 0.5 µL of this mixture was then spotted on the sample plate. Cationexchange resin beads (Bio-Rad, Hercules, CA) were also added to the sample plate to reduce the effects of alkali cations on the spectra.18 Calibration of the instrument was carried out using (dTp)18 and (pdT)5. Each spectrum is an average of 30-250 laser shots. Cleavage Reactions. Cleavage reactions were carried out by adding 0.5-1 µL of 2-iodoethanol (Aldrich, Milwaukee, WI) of (18) Nordhoff, E.; Ingendoh, A.; Cramer, R.; Overberg, A.; Stahl, B.; Karas, M.; Hillenkamp, F.; Crain, P. F. Rapid Commun. Mass Spectrom. 1992, 6, 771776.
various concentrations (see below) to the sample in the matrix before spotting on the MALDI sample plate. All reactions were conducted at 95 °C in a water bath and were quenched after the defined reaction time by immediately placing the sample in dry ice. RESULTS AND DISCUSSION In order to analyze the chemical reaction products using MALDI, the reaction products must be capable of cocrystallizing with the matrix upon evaporation of the solvent. As was recently reported by Dai et al.,19 the quality of the mass spectral results are highly dependent on the sample preparation method, which determines the extent of analyte incorporation within the matrix crystal. In the method of Nakamaye et al.,15 either 2,3-epoxy-1propanol (glycidol) or 2-iodoethanol was a suitable alkylating reagent for chemical cleavage of phosphorothioates. Glycidol has a boiling point of 62 °C/15 mm and is a slightly viscous solution. Upon addition of glycidol to the oligonucleotide/matrix solution, crystallization was not possible, and no suitable MALDI spectra were obtainable. The boiling point of 2-iodoethanol is 85 °C/25 (19) Dai, Y.; Whittal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500.
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mm, and upon addition to the oligonucleotide/matrix solution, crystallization was possible. Therefore, all experiments were performed using 2-iodoethanol. However, the quality of the mass spectral results are degraded by the addition of 2-iodoethanol, particularly for the larger, mixed-base oligonucleotides. The use of low concentrations of 2-iodoethanol (
