Improved Mass Analysis of Oligoribonucleotides by 13C, 15N Double

Feb 11, 2004 - Following purification and de- salting by reversed-phase HPLC, buffer exchange with. Microcon YM-3, and ethanol precipitation, electros...
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Anal. Chem. 2004, 76, 1804-1809

Improved Mass Analysis of Oligoribonucleotides by 13 C, 15N Double Depletion and Electrospray Ionization FT-ICR Mass Spectrometry Ying Xiong,† Kersten Schroeder,‡ Nancy L. Greenbaum,‡ Christopher L. Hendrickson,‡,§ and Alan G. Marshall*,‡,§

Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306, and Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005

13C, 15N

doubly depleted 32-ribonucleotide was synthesized enzymatically by in vitro transcription from nucleoside triphosphates isolated from E. coli grown in a minimal medium containing 12C, 14N-enriched glucose and ammonium sulfate. Following purification and desalting by reversed-phase HPLC, buffer exchange with Microcon YM-3, and ethanol precipitation, electrospray ionization Fourier transform ion cyclotron resonance mass spectra revealed greatly enhanced abundance of monoisotopic ions (by a factor of ∼100) and a narrower isotopic distribution with higher signal-to-noise ratio. The abrupt onset and high magnitude of the monoisotopic species promise to facilitate accurate mass measurement of RNA’s.

The past few years have witnessed revolutionary progress in the mass spectrometric determination of molecular weight of biomacromolecules, in general, and nucleic acids and their complexes in particular.1 For example, intact Escherichia coli 5s rRNA and yeast tRNAPhe desalted by HPLC have been detected, the latter to within a mass accuracy better than 10 ppm.2 Specific interactions between HIV Tat peptide and transactivation responsive element RNA have been shown to persist in the gas phase.3 In fact, automation has enabled the high-throughput screening of hundreds of thousands of potential RNA-binding drugs by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS).4 Determination of single nucleotide polymorphisms by mass measurement is now routine.5,6 Base composition of polymerase chain reaction (PCR) products can be derived from measured molecular weights and the complementary nature of †

Institute of Molecular Biophysics. Department of Chemistry and Biochemistry. § National High Magnetic Field Laboratory. (1) Limbach, P. A. Mass Spectrom. Rev. 1996, 15, 297-336. (2) Little, D. P.; Thannhauser, T. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2318-2322. (3) Sannes-Lowery, K. A.; Hu, P.; Mack, D. P.; Mei, H.; Loo, J. A. Anal. Chem. 1997, 69, 5130-5135. (4) Hofstadler, S. A.; Sannes-Lowery, K. A.; Crooke, S. T.; Ecker, D. J.; Sasmor, H.; Manalili, S.; Griffey, R. H. Anal. Chem. 1999, 71, 3436-3440. (5) Zhang, S.; Van Pelt, C. K.; Schultz, G. A. Anal. Chem. 2001, 73, 21172125. (6) Berger, B.; Holzl, G.; Oberacher, H.; Niederstatter, H.; Huber, C. G.; Parson, W. J. Chromatogr., B 2002, 782, 89-97. ‡

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double-stranded DNA,7 e.g., assignment of single-base substitutions in PCR products (89 base pairs and 114 base pairs).8 Furthermore, the locations of base substitutions, additions, and deletions can be determined by analyzing single-stranded PCR products produced by recombinant enzyme λ exonuclease that selectively digests one strand of a DNA duplex from a 5′phosphorylated terminus leaving the complementary strand intact.9 For RNA and DNA, mass accuracy to within ∼1 Da is required to distinguish between various nucleotides and their adducts or sequences: (a) uridine is 1 Da heavier than cytidine and 2 Da lighter than dihydrouridine; (b) methylated cytidine is 1 Da lighter than thymidine, methylated adeosine is 2 Da lighter than guanosine, and thiouridine is 2 Da heavier than thymidine; (c) an RNA with two potassium ions in place of protons (M + 2K - 2H) is 4 Da lighter than that by addition of one phosphate group (M + PO3H); (A + T) is 1 Da lighter than (G + C); etc. Finally, unit mass accuracy and narrower isotopic distribution10 are especially important for hydrogen-deuterium exchange experiments designed to identify solvent-exposed segments in a biomacromolecule, because each replacement of H by D adds 1 Da in mass.11 At first glance, achievement of such mass accuracy for RNA or DNA would appear to be easy. First, electrospray ionization (ESI12) can generate abundant multiply charged gas-phase unhydrated quasimolecular ions, (M - nH)n-, of mass-to-charge ratio, 500 e m/z e 3500, for DNA PCR products up to 500 base pairs (300 kDa),13 plasmid DNA (106 Da),14 and coliphage T4 DNA (100 MDa).15 ESI has even generated intact ribosomes detected by (7) Muddiman, D. C.; Anderson, G. A.; Hofstadler, S. A.; Smith, R. D. Anal. Chem 1997, 69, 1543-1549. (8) Muddiman, D. C.; Wunschel, D. S.; Liu, C.; Pasa-Tolic, L.; Fox, K. F.; Fox, A.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 3705-3712. (9) Null, A. P.; Hannis, J. C.; Muddiman, D. C. Analyst 2000, 125, 619-626. (10) Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S.; Guan, S.; Logan, T. M. J. Am. Chem. Soc. 1997, 119, 433-434. (11) Zhang, Z.; Li, W.; Logan, T. M.; Li, M.; Marshall, A. G. Protein Sci. 1997, 6, 2203-2217. (12) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (13) Muddiman, D. C.; Null, A. P.; Hannis, J. C. Rapid Commun. Mass Spectrom. 1999, 13, 1201-1204. (14) Cheng, X.; Camp, D. G., 2nd; Wu, Q.; Bakhtiar, R.; Springer, D. L.; Morris, B. J.; Bruce, J. E.; Anderson, G. A.; Edmonds, C. G.; Smith, R. D. Nucleic Acids Res. 1996, 24, 2183-2189. 10.1021/ac030299e CCC: $27.50

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time-of-flight (TOF) mass analysis.16 [For biomacromolecular assemblies larger than a few hundred thousand daltons, it may be impossible to eliminate solvent and salt adducts,16 so that the molecular weight of the “bare” complex is no longer accessible. Moreover, it is not possible to resolve charge states for ions higher than ∼1 MDa, so that one cannot distinguish mass and charge separately.] Second, the highest resolution mass analysis technique (FT-ICR MS)17 can routinely resolve a given mass spectral peak to within a few ppm for macromolecules up to several tens of thousands of daltons and an order of magnitude better than that in special cases.18 Nevertheless, even though proteins up to 112 kDa have been resolved to within 1 Da,19 it is generally difficult to determine mass unequivocally to within 1 Da for biomacromolecules larger than ∼15 kDa. The problem originates from the heavy isotopes of the common elements, primarily 13C and 15N. Although the natural abundances of 13C and 15N are low (1.11 and 0.36%, respectively), a protein or nucleic acid of tens of kilodaltons in mass contains hundreds to thousands of carbon and nitrogen atoms, producing a wide distribution in the natural abundance of molecules containing 1, 2, etc. 13C’s and/or 15N’s. Molecular weight of a biomacromolecule is thus typically reported as either the number-average mass (averaged over species containing from zero to all heavy atoms) or the “monoisotopic” mass (namely, all hydrogens are 1H, all carbons are 12C, all nitrogens are 14N, all oxygens are 16O, etc.). For molecules containing only C, H, N, O, S, and P atoms, the monoisotopic mass is the lowest of the possible isotopic combinations. As molecular weight increases, the average mass shifts (for proteins, by ∼1 Da for every 1.5 kDa in mass20,21) above the monoisotopic mass, and the isotopic distribution broadens to tens of daltons. Finally, the monoisotopic mass has a single isotopic composition, whereas all other nominal (i.e., nearest-integer) mass species consist of multiple “isobars” (e.g., molecules whose masses are ∼2 Da higher than the monoisotopic mass could contain 13C2, 13C15N, 15N2, 18O, 34S, etc.), each of whose exact masses differ by a few millidaltons. (In fact, even calculating the relative abundance at each nominal mass becomes cumbersome for biomolecules larger than ∼50 kDa.22) Thus, the monoisotopic molecule has the narrowest mass distribution and thus potentially highest mass accuracy. If the monoisotopic species can be detected, then the accurate mass can in principle be determined from the relative abundances of several of the lowest nominal mass species in the isotopic distribution.23 Unfortunately, for most biomolecules with molecular mass greater than ∼10 kDa, the monoisotopic relative abundance is so low as to be undetectable (10 kDa) produces a dominant monoisotopic peak with a compressed isotopic distribution, to yield higher signal-to-noise ratio and an unambiguous accurate monoisotopic mass. MATERIALS AND METHODS Materials. DNA templates (top strand, 5′ GGA TCC AAT ACG ACT CAC TAT AG 3′; bottom strand, 5′ GGG TGA TCT AAG CGC TTT CGC GCT GAT CAC CCT ATA GTG AGT CGT ATT GGA TCC 3′) were synthesized in the Biochemical Analysis Synthesis Sequencing Laboratory of the Department of Chemistry and Biochemistry, Florida State University. Glucose (99.9% 12C) and ammonium sulfate (99.99% 14N) were purchased from Isotec, Sigma-Aldrich (St. Louis, MO). Nuclease P1 (N8630), myokinase (M5520), pyruvate kinase (P9136), guanylate kinase (G7510 or G9385), and triethylammonium acetate (TEAA) were purchased from Sigma-Aldrich. NMP kinase was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Microcon YM-3 concentrators were purchased from Millipore Corp. (Bedford, MA). AG501-X8 mixed bed resin was purchased from Bio-Rad (Hercules, CA). Synthesis of 13C, 15N Doubly Depleted Ribonucleoside Triphosphates. In an adaptation of a previously published method,34 E. coli MY285 were grown in minimal medium M9 (6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 mM MgSO4, 1/2000 (v/v) Trace metal A (27 g/L FeCl3‚6H2O, 1.3 g/L ZnCl2, 2 g/L Na2MoO4‚2H2O, 2.5 g/L CaCl2‚2H2O, 2 g/L CoCl2‚6H2O, 1.3 g/L CuCl2‚2H2O, 3.3 g/L MnCl2‚4H2O, 0.5 g/L H3BO3, 1/10 (v/v) HCl), 5 mM (NH4)2SO4, 0.4% glucose) containing glucose (99.9% 12C) and ammonium sulfate (99.99% 14N). The total RNA fraction was isolated 34 and subsequently cleaved into ribonucleoside monophosphates by nuclease P1 (20 units/100 mg of RNA in a buffer of 0.1 mM ZnCl2, adjusted to pH 5.3 with 3 M NaAc, 37 °C, ∼2 h). Myokinase, pyruvate kinase, guanylate kinase, and NMP kinase were added to convert ribonucleoside monophosphates (rNMPs) to ribonucleoside triphosphates (rNTPs). Reaction products were subjected to Vydac ion chromatography HPLC (column 302IC4.6, 4.6-mm i.d., 250-mm length, 10-µm pore size). The mobile phase A was 25 mM aqueous NaH2PO4/Na2HPO4 (1:1 molar ratio), pH 2.8; phase B was 125 mM aqueous NaH2PO4/ Na2HPO4 (1:1 molar ratio), pH 2.9, with pH adjusted with acetic acid. The column was maintained at room temperature at a flow rate of 2.0 mL/min, and elution followed a linear gradient from 0 to 100% B in 23 min, monitored by optical absorbance at 260 nm. Figure 1 shows chromatograms before (top) and after (bottom) conversion from NMPs to NTPs, demonstrating high conversion efficiency. In Vitro Transcription of 13C, 15N Doubly Depleted Oligoribonucleotides. Transcription of the 32-ribonucleotide (5′ (34) Nikonowicz, E. P.; Sirr, A.; Legault, P.; Jucker, F. M.; Baer, L. M.; Pardi, A. Nucleic Acids Res. 1992, 20, 4507-4513.

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Figure 1. HPLC anion exchange chromatography of 13C, 15Ndepleted ribonucleoside monophosphates: before (top) and after (bottom) the in vitro phosphorylation reaction to form ribonucleoside triphosphates from the respective monophosphates.

GGG UGA UCA GCG CGA AAG CGC UUA GAU CAC CC 3′) was performed with synthetic DNA fragments containing a singlestranded template region and a double-stranded T7 promoter region.35 [For the transcription reactions in these experiments, we used templates with an extra six base pairs (on the 3′ terminus of the template strand and 5′ terminus of the “top” strand of the T7 promoter) to improve annealing efficiency. The T7 polymerase was isolated by Dr. Greenbaum’s laboratory according to the protocol of He et al.36 The last GC base pair where the top strand anneals to the template is the expected transcription start site. This start site is well documented in the literature35 and is supported by our measurements.] T7 RNA polymerase, 40 mM Mg2+, 4 mM GMP, 12.8 mM concentration of the total 13C, 15N doubly depleted NTPs, and 10× buffer (400 mM Tris-HCl, pH 8.0, 10 mM spermidine, 0.1% Triton, 300 mM dithiothreitol) were incubated at 37 °C for ∼9 h. Although “priming” the reaction with GMP is not strictly necessary, we used it to improve transcription yield. The reaction was quenched by addition of EDTA. The oligoribonucleotide product was examined by 20% denaturing PAGE immediately following transcription. Note that the oligoribonucleotide is thus equally depleted at each residue except for the 5′-terminal guanidine monophosphate, which was incorporated from commercial rGMP at natural abundance because use of isotopically depleted rGMP would not have significantly affected the final oligoribonucleotide isotopic distribution. Purification and Desalting. The 32-ribonucleotide was purified and partially desalted by reversed-phase HPLC (XTerra MS C18 column, 4.6-mm i.d., 50-mm length, 2.5-µm pore size) from Waters (Milford, MA). The mobile phase A was 1.5% acetonitrile/ water, 100 mM TEAA, pH 7.0; phase B was 50% acetonitrile/water. The column was heated to 55 °C. Elution followed a linear gradient from 5 to 25% B in 30 min at 1.0 mL/min and was monitored by UV absorbance at 260 nm. The collected fraction was loaded to the column one more time. The final collection was lyophilized and checked by 20% denaturing PAGE to make sure that the correct oligoribonucleotides were collected. Afterward, Microcon (35) Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C. Nucleic Acids Res. 1987, 15, 8783-8798. (36) He, B.; Rong, M.; Lyakhov, D.; Gartenstein, H.; Diaz, G.; Castagna, R.; McAllister, W. T.; Durbin, R. K. Protein Expression Purif. 1997, 9, 142151.

YM-3 (MWCO 3000, Bedford, MA) was applied to change the buffer to 1.0 mM ammonium phosphate dibasic. Glycerol was removed from Microcon filters in advance by soaking them in 10 mM NaOH and washing with pure water. A dilution of at least 1:105 of the original buffer is needed to avoid excessive salt adduct formation during electrospray ionization. In addition, multiple ethanol precipitations with 1/3 (v/v)10 M ammonium acetate at -20 °C overnight were needed to reduce the salt concentration further. Microelectrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Mass analysis was performed with a home-built FT-ICR mass spectrometer equipped with a 22-cm-diameter, horizontal-bore 9.4-T magnet (Oxford Corp., Oxney Mead, England).37 13C, 15N doubly depleted oligoribonucleotide was suspended in 50% acetonitrile/water, 0.5% NH3/ H2O. Deionized water was treated by AG501-X8 mixed-bed resin before use. Glassware containers were avoided during sample preparation. Data were collected and processed with a modular ICR data acquisition system (MIDAS).38,39 Negative ions were generated from a microelectrospray40 source equipped with a 50µm-i.d. fused-silica microelectrospray needle. Samples were infused at a flow rate of 500 nL/min. Typical ESI conditions were as follows: needle voltage, -2.0 kV; tube lens, -370 V; and heated capillary current, 3.5 A. Ions were accumulated external to the magnet41 in a linear octopole ion trap (14 cm long) for 15 s and transferred through rf-only multipoles to a 10-cm-diameter, 30cm-long open cylindrical Penning ion trap. Multipoles were operated at 1.5 MHz at a peak-to-peak rf amplitude of 170 V. Broadband frequency-sweep (“chirp”) dipolar excitation42 (57 kHz-0.32 MHz at a sweep rate of 150 Hz/µs and peak-topeak amplitude, 190 V) was followed by direct-mode image current detection that yielded 512 kword time-domain data. The timedomain data were baseline-corrected and Hanning-apodized, followed by a single zero-fill before fast Fourier transformation and magnitude calculation. Frequency was converted to mass-tocharge ratio (m/z) by the quadrupolar electric trapping potential approximation43,44 to generate a m/z spectrum. Mass spectra were internally calibrated from the measured ICR frequencies of ES tuning mix (Agilent Technologies, Palo Alto, CA) (m/z 734.007 26, 1033.988 10, 1633.949 77). RESULTS AND DISCUSSION Removal of Salt Adducts. Most of the negatively charged phosphates in a polynucleotide are neutralized by protons. (37) Senko, M. W.; Hendrickson, C. L.; Pasa-Tolic, L.; Marto, J. A.; White, F. M.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (38) Senko, M. W.; Canterbury, J. D.; Guan, S.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1839-1844. (39) Blakney, G. T.; van der Rest, G.; Johnson, J. R.; Freitas, M. A.; Drader, J. J.; Shi, S. D.-H.; Hendrickson, C. L.; Kelleher, N. L.; Marshall, A. G. Further Improvements to the MIDAS Data Station for FT-ICR Mass Spectrometry, In Proc. 49th Am. Soc. Mass Spectrom. Conf. Mass Spectrom., Allied Top.; Am. Soc. Mass Spectrom.: Chicago, IL, 2001. (40) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (41) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (42) Marshall, A. G.; Roe, D. C. J. Chem. Phys. 1980, 73, 1581-1590. (43) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 2744-2748. (44) Shi, S. D.-H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, 195/196, 591-598.

However, nonvolatile cations such as sodium or potassium can replace one or more protons, leading to additional heterogeneity in mass with corresponding reduction in the relative abundance at any one nominal mass and thus reduced signal-to-noise ratio and reduced mass accuracy. It is therefore desirable to remove those cations in solution before mass spectrometric analysis and to add chelating agents or volatile cations to the sample to suppress binding of Na+ and K+ cations to target nucleic acids during the electrospray process. To remove Na+ and K+ in the original sample, we employed reversed-phase HPLC with TEAA additives in the mobile phase, microcons to remove Na+ and K+ in the buffer, and ethanol precipitation in the presence of ammonium acetate. The chelation approach was first proposed and demonstrated by Grotjahn.45 Subsequently, Limbach added cyclohexyldiaminetetraacetic acid to remove divalent metal ions and triethylamine to remove monovalent cations.46 Organic bases with solution pKb values from 5.5 to 11.5 and gas-phase proton affinities from 213 to 232 kcal/ mol have also been examined.47 Finally, on-line microdialysis48-51 has proved to be effective for desalting of nucleic acids. Here, treatment of deionized water with AG501-X8 mixed-bed resin proved effective in eliminating trace cations. Ammonium hydroxide added to the acetonitrile electrospray solvent was found to be more effective than other additives. Natural-Abundance (98.9% 12C and 99.64% 14N) 32Ribonucleotide. Success in eliminating Na+ and K+ adducts is demonstrated by the ESI FT-ICR mass spectrum shown in Figure 2, top. The isotopic distribution for the natural-abundance 32ribonucleotide is nearly symmetric and spreads over ∼12 Da, with very low levels of singly sodiated or potassiated species. The observed relative abundance of the monoisotopic ions is ∼2.3% (vs 7% calculated from the chemical formula for the neutral molecule C306H381N127O222P32). (Observed FT-ICR abundances of low-abundance ions can be suppressed by high-abundance ions in the same spectrum, due to Coulomb forces in the orbiting ion packets.52) The monoisotopic mass of the neutral molecule, is 10 376.48 ( 0.31 Da, i.e., ∼7.7 ppm higher than the 10 376.40 Da calculated from the known nucleotide sequence. The standard deviations for mass measurements for natural abundance and doubly depleted 32-mer were calculated from the seven and nine most abundant charge states, respectively. For each charge state, the monoisotopic nominal mass was established by matching the experimental and calculated isotope distributions. The monoisotopic mass was then determined from the mass of the monoisotopic peak. The monoisotopic neutral masses were then weighted according to the relative abundances of those charge states. (45) Grotjahn, L.; Blocker, H.; Frank, R. Biomed. Mass Spectrom. 1985, 12, 514524. (46) Limbach, P. A.; Crain, P. F.; A., M. J. J. Am. Soc. Mass Spectrom. 1995, 6, 27-39. (47) Greig, M.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97102. (48) Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359363. (49) Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893-4897. (50) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325. (51) Liu, C.; Wu, Q.; Harms, A. C.; Smith, R. D. Anal. Chem. 1996, 68, 32953299. (52) Mitchell, D. W.; Smith, R. D. Phys. Rev. E 1995, 52, 4366-4386.

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Figure 2. FT-ICR mass spectra of the oligoribonucleotide 32-mer. Top: natural abundance isotopic distribution (∼98.89% 12C, ∼99.63% 14N). Bottom: isotopic distribution for the same oligoribonucleotide produced from isotopically depleted rNTPs (∼99.9% 12C, 99.99% 14N). Based on the observed isotopic distribution, the actual isotopic composition of the doubly depleted oligoribonucleotide reflects that of the medium to within experimental error (99.91% 12C and 99.98% 14N).

13C, 15N

Doubly Depleted (99.9% 12C and 99.99% 14N) 32-Ribonucleotide. In contrast, the isotopic distribution for the 13C, 15N doubly depleted oligonucleotide (Figure 2, bottom) is asymmetric and narrower than that for the natural-abundance oligoribonucleotide. Moreover, the monoisotopic peak is now highest in abundance and easily recognized by its sudden onset. The molecular weight of the neutral molecule (abundanceweighted average over the isotopic distributions for all observed charge states) is thus determined unambiguously as 10 376.33 ( 0.13 Da, i.e., 6.7 ppm less than 10 376.40 Da calculated from the molecular formula. In this case, the molecular weight accuracy is comparable to that calculated for the weighted average in the natural-abundance oligoribonucleotide, but only because the highest magnitude peak in the natural-abundance isotopic distribution (see Figure 2, top) was the correct onesif we had chosen the next-nearest peak, the mass would have been off by a full dalton. There is no such ambiguity for the doubly depleted oligoribonucleotide (Figure 2, bottom), because the monoisotopic peak is apparent from its sudden onset in the mass spectrum. Predicted Improvement in Detection of Monoisotopic Species by Use of 13C, 15N Double Depletion. Consider a protein whose amino acid composition is the same as the average amino acid composition of all proteins in the SwissProt database, namely, C4.9384H7.7583N1.3577O1.4773S0.0417 per “averagine” amino acid residue.24 The highest mass at which the monoisotopic abundance is at least 1% of that of the most abundant nominal mass species increases from ∼10 (naturally abundant 12C and 14N) to ∼53 (99.95% 12C), 64 (99.9% 12C and 99.99% 14N), and ∼100 kDa (99.99% 12C and 99.99% 14N).10,24 The same approach can be applied to DNA or RNA, for which the elemental composition of an average nucleotide residue (i.e., one nucleotide minus H2O due to the formation of the phosphodiester linkage), “averabaseine”, is C9.75H12.25N3.75O6P1 for DNA25 and C9.5H11.75N3.75O7P1 for RNA, based on random occurrence of each of the four DNA or RNA bases. [The overall relative 1808 Analytical Chemistry, Vol. 76, No. 6, March 15, 2004

Figure 3. Monoisotopic ion relative abundance for RNA containing equal numbers of all four bases, as a function of molecular mass for natural abundance and each of three levels of isotopic depletion of 13C and 15N. Note that 13C and 15N isotopic depletion greatly increases the monoisotopic peak abundance at high mass, permitting more direct and reliable molecular weight determination. (The corresponding plot for DNA is essentially identical.)

abundance of (G + C) in the human genome is ∼40%;53 and the transcription of various genes is variable: ergo, there is no simple way to estimate the actual average relative abundances of the ribonucleotides.] Thus, the mass of RNA consisting of n “averabaseines” would be n times the mass of one “averabaseine” plus the mass of H2O (due to the extra H and OH at the 3′ and 5′ termini). For example, from a given experimental ribonucleic acid mass, Figure 3 shows that the highest mass at which the monoisotopic relative abundance exceeds 1% increases from 15 (natural-abundance 12C and 14N) to 60 (99.95% 12C), 78 (99.9% 12C and 99.99% 14N), and 110 kDa (99.99% 12C and 99.99% 14N). Similar patterns are found for DNA, except for slight differences due to the deoxy- versus oxypentose and the replacement of T by U in RNA compared to DNA. The theoretical effects of increasing enrichment of 12C and 14N in proteins and DNA have been discussed extensively by Zubarev and Demirev.25 In particular, they point out that although isotopic depletion narrows the isotopic distribution for biomacromolecules (typically ∼10 ppm for natural abundance), the degree of depletion adds uncertainty to the experimental mass determination (if the monoisotopic abundance is too low to measure). In that respect, it is encouraging to note that the isotopic distribution observed experimentally in Figure 2 (bottom) is consistent with the (known) degrees of depletion of 13C and 15N in the glucose and ammonium sulfate used to produce the oligoribonucleotide. They also note that extreme isotopic depletion does not significantly improve mass accuracy; however, in practice, the degree of isotopic depletion is limited by cost before any theoretical limits are reached. Of course, the main advantage of double depletion, as noted above, is the sudden onset of the monoisotopic mass spectral peak, so that there is no ambiguity about assigning the correct molecular mass to the nearest dalton. Figure 4 demonstrates graphically the improvement produced by 13C,15N double depletion in distinguishing two oligoribonucleotide 32-mers differing in mass by 1 Da (i.e., by replacement of a single U by C). Specifically, double depletion increases the rms standard deviation between the two RNA isotopic distributions by a factor of 5.2. (53) Ikemura, T.; Wada, K. Nucleic Acids Res. 1991, 19, 4333-4339.

Figure 4. Simulated isotopic distributions, broadened to a resolution of ∼1 Da, for oligoribonucleotide 32-mers differing by substitution of a single U (left) for C (right) (i.e., a 1-Da increase in molecular mass). Top: natural abundance. Bottom: double depletion in 13C (99.9% 12C) and 15N (99.99% 14N). Double depletion increases the rms standard deviation between the two oligoribonucleotides isotopic distributions by more than a factor of 5 (see text).

Finally, because doubly depleted rNMPs and rNTPs were not commercially available, we were forced to generate them from bacteria grown from doubly depleted nutrients. If and when doubly depleted ribo- and deoxyribonucleoside mono- and triphosphates become available commercially, the effort to produce doubly depleted RNA or DNA will be greatly reduced. CONCLUSION The results in Figures 2 and 3 demonstrate that the experimental and theoretical advantages of 13C, 15N double depletion are approximately comparable for RNA (and theoretically for

DNA) as previously found for proteins.10,28,29 In particular, the first direct monoisotopic mass measurement for RNA of g10 kDa is demonstrated. As noted above, direct observation of the monoisotopic species (and thus the certain knowledge of mass to the nearest dalton) should make it possible to distinguish between additional bases, as well as to identify nucleoside modifications and various adducts. With that knowledge, one may go on to use the accurate mass to define nucleic acid composition. Finally, the effects of double depletion are most dramatic for ultrahigh-resolution FT-ICR mass spectra, at mass resolving power sufficient to resolve individual nominal masses (so that charge state of a multiply charged ion may be determined directly24). However, double depletion benefits lower resolution mass analyzers as well, by reducing the width of the isotopic distribution (for better resolution of bare and adducted DNA or RNA and improved signal-to-noise ratio). Optimal exploitation of those advantages will require extensive prior desalting to remove Na and K adducts. ACKNOWLEDGMENT We thank Mark R. Emmett, Kristina Håkansson, Michael J. Chalmers; John P. Quinn, Zhigang Wu, Melinda McFarland, Laura Jane Phelps, Meredith Newby Lambert, Hank Henricks, and Umesh Goli for their generous and helpful advice. This work was supported by the NSF National High-Field FT-ICR Mass Spectrometry Facility (CHE 99-09502), Florida State University, and the National High Magnetic Field Laboratory at Tallahassee, FL.

Received for review August 12, 2003. Accepted January 6, 2004. AC030299E

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