Detection of the Common RNA Nucleoside Pseudouridine in Mixtures

Milwaukee, WI) and 50 μL of acetonitrile (ACN) (J. T. Baker, Phillipsburg, NJ). ... 11γ, UAACUAΨAACG) were synthesized at Dharmacon Research (B...
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Anal. Chem. 2005, 77, 4687-4697

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Detection of the Common RNA Nucleoside Pseudouridine in Mixtures of Oligonucleotides by Mass Spectrometry Steven C. Pomerantz† and James A. McCloskey*,†,‡

Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 84112

Pseudouridine, an isomer of uridine, is probably the most common of many posttranscriptional RNA modifications found in nature. Although mass spectrometry has become widely used in the characterization of modified nucleic acids, its application to the recognition and sequence placement of pseudouridine has not been straightforward, particularly in the case of complex mixtures such as those resulting from selective enzymatic hydrolysis of RNA into oligonucleotides. We report results of a study of the characteristic dissociation reactions of pseudouridinecontaining oligonucleotides following ionization by electrospray and use of those pathways in an LC/MS-based method applicable to direct analysis of RNase digests of RNA. As a consequence of the C-C (rather than C-N) glycosidic bond of pseudouridine, the otherwise common dissociation paths involving base loss do not occur, resulting in characteristic formation of a set of low-mass negative ions containing the intact glycosidic bond (m/z 225, 207, 189, 165, 164, 139), which permit recognition of pseudouridine-containing oligonucleotides. Those components can subsequently be subjected to sequence analysis by MS/MS, in which enhancement of selective sequence-determining ions (a-, w-, y-types), and absence of a - base ions, are observed at the site of pseudouridylation. Also, selected reaction pathways can be monitored in the LC/MS/MS analysis that are indicative of pseudouridine at the 5′ terminus (m/z 225 f 165), internal positions (m/z 207 f 164), and in the RNase T1-derived product ΨpGp (m/z 668 f 207) arising from the RNA sequence ...GΨG... These procedures can be effectively integrated into an existing suite of LC/ESI-MS-based methods designed for the analysis of posttranscriptionally modified sites in RNA. * Corresponding author. Phone: (801) 581 5581. Fax: (801) 581 7532. E-mail: [email protected]. † Department of Medicinal Chemistry. ‡ Department of Biochemistry. 10.1021/ac058023p CCC: $30.25 Published on Web 06/22/2005

© 2005 American Chemical Society

RNA is endowed with an exceptional number and structural diversity of posttranscriptional modifications used for manifold purposes, which are biosynthesized by an array of genomically encoded enzymes.1-3 In general, these modifications serve to finetune and regulate RNA structure and hence its myriad functional roles. Many, if not most, of these modifications tend to cluster at sites of functional biological importance, e.g., the anticodon loop of tRNA4 or the peptidyl transferase center of large subunit rRNA5 and the decoding region of the small ribosomal subunit.6 Pseudouridine (Ψ; see Figure 2, m/z 243 for structure) is the single most prevalent7 of the ∼102 known modified nucleosides that occur in RNA;8 see also http://medlib.med.utah.edu/RNAmods/. It has been found in most types of RNA associated with protein synthesis, including tRNA,9 tmRNA,10 and ribosomal9 and small nucleolar RNAs.11 Pseudouridine does not appear to have one overall role;12 instead, it has been implicated in such diverse functions as RNA structure stabilization, through base stacking and iminoproton-H2O coordination,13,14 codon-anticodon interac(1) Maden, B. E. H. Prog. Nucleic Acids Res. Mol. Biol. 1990, 39, 241-303. (2) Grosjean, H., Benne, R., Eds. Modification and Editing of RNA; ASM Press: Washington, DC, 1998. (3) Agris, P. F. Nucleic Acids Res. 2004, 32, 223-238. (4) Bjo ¨rk, G.; Ericson, J. U.; Gustafsson, C. E. D.; Hagervall, T. G.; Jo¨nsson, Y. H.; Wikstro ¨m, P. M. Annu. Rev. Biochem. 1987, 56, 263-287. (5) Sirum-Connolly, K.; Peltier, J. M.; Crain, P. F.; McCloskey, J. A.; Mason, T. L. Biochimie 1995, 77, 30-39. (6) Mueller, F.; Brimacombe, R. J. Mol. Biol. 1997, 271, 524-544. (7) Maden, B. E. H.; Hughes, J. M. X. Chromosoma 1997, 105, 391-400. (8) Rozenski, J.; Crain, P. F.; McCloskey, J. A. Nucleic Acids Res. 1999, 27, 196-197. (9) Dunn, D. B. Biochim. Biophys. Acta 1959, 34, 286-287. (10) Felden, B.; Hanawa, K.; Atkins, J. F.; Himeno, H.; Muto, A.; Gesteland, R. F.; McCloskey, J. A.; Crain, P. F. EMBO J. 1998, 17, 3188-3196. (11) Reddy, R.; Ro-Choi, T. S.; Henning, D.; Shibata, H.; Choi, Y. C.; Busch, H. J. Biol. Chem. 1972, 247, 7245-7250. (12) Charette, M.; Gray, M. W. IUBMB Life 2000, 49, 341-351. (13) Arnez, J. G.; Steitz, T. A. Biochemistry 1994, 33, 7560-7567. (14) Davis, D. R. Nucleic Acids Res. 1995, 23, 5020-5026.

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tions,15 and ribosome assembly.16 Pseudouridine is unique among all known RNA nucleosides in that it does not manifest a mass shift upon posttranscriptional formation that would thus make it in principle detectable by mass spectrometry. Most other modifications can be unequivocally detected by mass measurement alone to determine the nature of the modification, while sequence placement within the oligonucleotide can often be accomplished by tandem MS. Earlier, the principal chemically based methods for the detection of Ψ had been analysis of the nucleoside constituents of RNA following total hydrolysis, e.g., by combined LC/MS17 or reversed-phase HPLC.18 The most widely used contemporary methods for detection and sequence placement of Ψ in RNA are variations of the procedures introduced by Ofengand,19 in which N-3 of Ψ is irreversibly derivatized using the reagent N-cyclohexyl-N′-β-(4methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), which causes a characteristic gel stop upon reverse transcriptase (RT) extension of appropriately designed and synthesized radiolabeled primers.20 This method offers subpicomole sensitivity but suffers from two principal limitations. First, Ψ residues can be detected only in RNA downstream regions intentionally probed by preselected RT primers (and thus excluding 3′-terminal portions of the RNA). Second, care must be taken to ensure complete deblocking of reversibly derivatized guanosine, inosine, and uridine in the initial reaction sequence, to avoid the serious problem of false positives.21 The sequence coverage limitation of the CMC method can be effectively avoided by mass measurement of oligonucleotides derived from the CMC-derivatized RNA, in which a 252-Da mass shift due to the Ψ derivative allows differentiation of Ψ from U.22 Treatment of the RNA by RNase T1, collection of fractions, and mass analysis by MALDI-MS is then followed by timed exonuclease digestion with product monitoring by mass spectrometry for sequence placement. More recently, Mengel-Jørgensen and Kirpekar developed an analogous mass shift method in which pseudouridines (and 4-thiouridine if present) in the RNA are selectively cyanoethylated using acrylonitrile, resulting in a 52Da mass shift compared with uridines.23 Sequencing of oligonucleotides deemed to be of interest is then carried out using MALDI-MS, following RNase treatment and mass measurement of the resulting oligonucleotides. The method presented here is designed for detection and sequence placement of pseudouridine using LC/ESI-MS and LC/ ESI-MS/MS and is intended for integration into a suite of LC/ MS-based protocols designed for application to mixtures of RNA oligonucleotides.24 Emphasis is placed on the applicability to (15) Davis, D. R.; Poulter, C. D. Biochemistry 1991, 30, 4223-4231. (16) Cunningham, P. R.; Richard, R. B.; Weitzmann, C. J.; Nurse, K.; Ofengand, J. Biochimie 1991, 73, 789-796. (17) Edmonds, C. G.; Vestal, M. L.; McCloskey, J. A. Nucleic Acids Res. 1985, 13, 8197-8206. (18) Gehrke, C. W.; Kuo, K. C. In Chromatography and Identification of Nucleosides, part A; Gehrke, C. W., Kuo, K. C., Eds.; Journal of Chromatography Library Vol. 45A; Elsevier: New York, 1990; pp A3-A64. (19) Bakin, A.; Ofengand, J. Biochemistry 1993, 32, 9754-9762. (20) Ofengand, J.; Del Campo, M.; Kaya, Y. Methods 2001, 25, 365-373. (21) Del Campo, M.; Recinos, C.; Yanez, G.; Pomerantz, S. C.; Guymon, R.; Crain, P. F.; McCloskey, J. A.; Ofengand, J. RNA 2005, 11, 210-219. (22) Patteson, K. G.; Rodicio, L. P.; Limbach, P. A. Nucleic Acids Res. 2001, 29, e49-e59. (23) Mengel-Jørgensen, J.; Kirpekar, F. Nucleic Acids Res. 2002, 30, e135.

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complex mixtures, generally avoiding intermediate steps involving isolation of oligonucleotide fractions. The fragmentation reactions described here that are employed for the selective identification of pseudouridine (which possesses a C-C glycosidic bond) differ from those of the isomer uridine, which has a C-N glycosidic bond, and take advantage of the absence of common glycosidic bond dissociation reactions, which are channeled into other dissociation pathways.

EXPERIMENTAL SECTION Oligonucleotide Synthesis and Preparation. All oligonucleotides, except for the members of the isomeric 11-mer series, were synthesized at the 1-µmol scale on an Applied Biosystems (Foster City, CA) model 394 instrument, using phosphoramidite coupling chemistry with standard cycles and conditions, in the DNA/ Peptide Synthesis Facility at the University of Utah, and were delivered as 2′-hydroxy- and base-protected (2′-OH, tert-butyldimethylsilyl (t-BDMS); Cyt, acetyl; Gua, phenoxyacetyl; Ade, isopropylphenoxyacetyl) products. Oligonucleotides were basedeprotected and cleaved from the solid support in this laboratory by immersion in 4 mL of concentrated ammonium hydroxide in 25% (v/v) ethanol for 2 h. The solvent was decanted and a fresh aliquot of 2 mL added for 1 h. The supernatants were pooled, incubated at 55 °C for 6 h to effect removal of the base-protecting groups, and then evaporated to dryness. Removal of the t-BDMS protecting groups on the 2′-hydroxyl of the ribose moieties was achieved by vigorous agitation for ∼16 h in 500 µL of triethylamine trihydrofluoride25 (Aldrich, Milwaukee, WI) and 50 µL of acetonitrile (ACN) (J. T. Baker, Phillipsburg, NJ). The reaction was quenched by addition of 500 µL of H2O and evaporated under reduced pressure in a SpeedVac (Savant, Farmingdale, NY). Samples were reconstituted in 200-300 µL of H2O for HPLC desalting and fractionation of the failure sequences. The β- and γ-substituted 11-mers (11β, UAACΨAUAACG; 11γ, UAACUAΨAACG) were synthesized at Dharmacon Research (Boulder, CO) on a 1-µmol scale and deblocked in this laboratory. Removal of the 2′-bis(acetoxyethoxy) methyl ether protecting groups was accomplished by dissolving a 250-nmol aliquot in 400 µL of 100 mM acetic acid titrated to pH 3.8 with tetramethylethylenediamine. The reaction mixture was incubated at 60 °C for 30 min and immediately purified by HPLC as described below. The R isomer (11R, ΨAACUAUAACG) was prepared as follows. The 19-mer GGCCGΨAACUAUAACGGUC was synthesized at Dharmacon. After deprotection as described above, a 50-nmol aliquot was digested with 5000 units of ribonuclease T1 (Ambion, Austin, TX) in 148 µL of 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) and 0.5 mM ethylenediaminetetraacetic acid (EDTA). After HPLC fractionation as described below, the 11-mer ΨAACUAUAACGp was dephosphorylated with 500 units of calf intestinal phosphatase (New England Biolabs, Beverly, MA) in 138 µL of a buffer consisting of 50 mM sodium chloride, 10 mM Tris-HCl, 10 mM magnesium chloride, and 1 mM dithiothreitol. After incubation at 37 °C for 15 min, the (24) Crain, P. F.; Ruffner, D. E.; Ho, Y.; Qiu, F.; Rozenski, J.; McCloskey, J. A. In Mass Spectrometry in Biology and Medicine; Burlingame, A. L., Carr, S. A., Baldwin, M. A., Eds.; Humana Press: Totowa, NJ, 1999; pp 531-551. (25) Gasparutto, D.; Livache, T.; Bazin, H.; Duplaa, A. M.; Guy, A.; Khorlin, A.; Molko, D.; Roget, A.; Teoule, R. Nucleic Acids Res. 1992, 20, 5159-5166.

dephosphorylated product was immediately isolated by HPLC to prevent degradation of the sample by nuclease contamination of the phosphatase. RNase T1 Digestion of Escherichia coli tRNA2Tyr. An aliquot containing 120 pmol of the tRNA (Sigma, St. Louis, MO) was diluted from a concentrated stock solution to a volume of 17 µL, Tris-EDTA buffer (100 mM Tris, pH 7.5; 10 mM EDTA; 2 µL) was added, the sample was thermally denatured for 2 min at 100 °C, and then plunged into an ice/water bath. Enzymatic digestion of the sample was accomplished by addition of 50 units of RNase T1 (Ambion, Austin, TX) in 1 µL of Tris-EDTA buffer and incubation of the sample in a 37 °C water bath for 30 min. RNase U2 Digestion of E. coli 16S rRNA. An aliquot (250 pmol) of E. coli 16S rRNA (isolated in this laboratory by E. Bruenger) in 25 µL of 10 mM ammonium acetate (pH 4.5) was denatured at 100 °C for 2 min and then digested with 30 units of RNase U2 (Pharmacia, Piscataway, NJ) for 15 min at 60 °C. An additional 30 units of enzyme was added, and digestion continued for 15 min. The reaction was quenched in an ice bath, and a 30pmol aliquot was immediately analyzed by LC/MS and LC/MS/ MS (same experiment) to determine which oligonucleotides contained Ψ. A 100-pmol aliquot was then subject to LC/MS/MS with timed precursor ion selection for the sequence determination of the liberated oligonucleotides which were demonstrated to contain Ψ in the previous experiment. Oligonucleotide Purification. Isolation and purification of all synthetic samples was performed by gradient RP-HPLC on a Beckman (Fullerton, CA) model 126 chromatograph. The column was a 4.6 × 250 mm LC-18S (Supelco, Bellefonte, PA) protected by a 2.1 × 10 mm precolumn of the same type. Buffer A was 25 mM triethylammonium bicarbonate, pH 6.0, and buffer B was 40:60 (v/v) ACN/water. The gradient was 0-50% B at 1%/ min, and the liquid flow was 1 mL/min. Detection at 280 nm was accomplished with a Waters (Milford, MA) model 440 UV detector, and fraction collection was performed manually. Collected fractions were dried, reconstituted, and quantified on a Perkin-Elmer (Norwalk, CT) Lambda 3 spectrophotometer at 260 nm. Infusion Mass Spectrometry. Direct sample infusion experiments were performed on a Sciex (Concord, ON, Canada) API III+ triple quadrupole instrument fitted with the articulated IonSpray source. Synthetic samples were dissolved at high concentration in water to achieve final concentrations of 5-20 pmol/µL in water/methanol (5:95 or 10:90), water/2-propanol (50: 50), or 2 mM ammonium acetate/ACN (50:50). Samples were infused via syringe pump (Model 22, Harvard Apparatus, Holliston, MA). Mass spectra were acquired in negative ion mode, with a step size of 0.1 u, and dwell times at each step ranging from 0.5 to 5 ms. Multichannel acquisition mode was employed in all cases, with 10-100 spectra summed for improved S/N ratios and to minimize temporal variations in performance. Resolution in the first and third quadrupoles was sufficient to resolve the isotope peaks of a triply charged ion at m/z 802 in primary mass spectra. The resolution in Q1 was relaxed to improve abundance in product ion spectra, and the resolution was reduced in Q1 and Q3, when necessary, for acquisition of precursor ion spectra. Negative ion electrospray ionization was accomplished at 1.5-2 µL/min using

the standard Sciex nebulizer with a 190 µm o.d. × 75 µm i.d. fusedsilica transfer line, zero grade air for the nebulizing gas, and nitrogen for the counterflow drying gas (∼600-1200 mL/min). Alternatively, nanospray ionization at 100-300 nL/min was performed by a system designed by the authors that employed narrow-bore fused-silica capillary tips. Needle voltages varied from 1100 to 1800 V, depending on the flow rate and orifice diameter of the tip being used. The high voltage for ionization was introduced via a liquid junction interface in a stainless steel union (Valco; Houston, TX) between the PEEK transfer line (640 µm o.d. × 64 µm i.d., Upchurch Scientific, Oak Harbor, WA) and the emitter. Nanospray capillary tips were purchased from New Objective (Cambridge, MA) in 360 µm o.d. × 75 µm i.d. with 15µm tip diameters, or 360 × 20 × 10 µm geometry. The capillary was mounted in the liquid junction interface with PEEK tubing sleeves and nuts. Some nanospray capillaries were also fabricated in the laboratory. No nebulization gas was employed for nanospray ionization experiments, and the flow rate of the counterflow drying gas was reduced to ∼300 mL/min. In all cases, the ion sampling skimmer cone and entrance to the mass spectrometer was maintained at 60 °C. Tandem mass spectra of all types were acquired with argon as the collision gas at a thickness of ∼275 × 1013 atoms/cm2 (∼5.5 mTorr). Directly Combined LC/MS and LC/MS/MS. Combined LC/MS and LC/MS/MS experiments were performed on a Micromass (Wythenshawe, U.K.) Quattro II triple quadrupole instrument with either the original coaxial or the Z-spray electrospray ion source coupled to a Hewlett-Packard (Palo Alto, CA) model 1090 liquid chromatograph with diode array detector. The combined instrument was operated under the control of the Micromass MassLynx MS data system. Buffer A was 400 mM hexafluoro-2-propanol (HFIP; J. T. Baker, Phillipsburg, NJ), titrated to pH 7.0 with triethylamine. Buffer B was 400 mM HFIP, pH 7.0, in 50% aqueous methanol.26 The gradient was linear at 2%/min from 0 to 100% B, with a 5-min hold at 100% B to ensure complete elution of the samples. UV spectra were recorded every 0.75 s from 210 to 320 nm. Separations were performed at 40 °C on a Supelco LC-18S column (1 × 300 mm, 5 µm) protected by an Optimize Technologies (Oregon City, OR) Opti-Guard C18 precolumn (1 × 15 mm). The entire effluent (60 µL/min) of the LC was coupled to the electrospray probe via PEEK tubing (1/16 × 0.005 in. i.d., Upchurch Scientific). Negative ion mass spectra were recorded in continuum mode from m/z 300 to 1500 in 1.5 s with the ion sampling cone set to 50-60 (coaxial source) or 30-40 V (Z-spray source). An elevated nozzle-skimmer voltage (“base release”) was used for data acquisition in centroid mode from m/z 100 to 300 in 0.5 s with the cone set at 110 V. The presence of Ψ was detected by a reaction monitoring function that recorded the transition from m/z 207 to 164 with a dwell time of 0.4 s at a collision energy of 12 eV, a cone voltage of 200 V, and ∼3 × 10-3 mbar argon in the collision cell. The total MS cycle time was 2.5 s, preserving chromatographic fidelity by acquiring ∼10 spectra/ LC peak. LC/MS/MS sequencing of oligonucleotides from an RNase U2 digest of E. coli 16S rRNA was performed on a Micromass Q-Tof 2 instrument connected to a Waters CapLC chromatograph. (26) Apffel, A.; Chakel, J. A.; Fischer, S.; Lichtenwalter, K.; Hancock, W. S. Anal. Chem. 1997, 69, 1320-1325.

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The chromatography employed the same HFIP buffer system described above, which was delivered at a flow rate of 15 µL/min to a Phenomenex (Torrance, CA) Luna C18(2) column (0.5 × 150 mm, 3 µm) protected by a LC Packings (Sunnyvale, CA) µ-Precolumn cartridge (PepMap C18, 0.3 × 5 mm, 5 µm). Cation adduct formation was suppressed by the postcolumn addition of a solution of 5 mM CDTA and 1 mM imidazole delivered at 1 µL/min by a Harvard Apparatus model 22 syringe pump through an Upchurch PEEK tee. Precursor ion masses and retention times were determined from a preliminary LC/MS analysis. The 3- and 4charge-state ions of the oligonucleotide CUCCGΨG>p were both subjected to 60-eV collisions (Elab), while the 6- and 4- chargestate ions of the oligonucleotide CUCCGΨGCCA>p were subjected to collisions of 90 and 88 eV, respectively. Product ion mass spectra were recorded from m/z 100 to 1990.

RESULTS AND DISCUSSION Dissociation Chemistry of Ψ-Containing Oligonucleotides. Identification of Characteristic Fragment Ions from Pseudouridine in Oligonucleotides. Initial investigations were carried out on a series of homopolymers, d(U4)p, U4p, and Ψ4p, to clearly establish the identities of Ψ-related ions. These oligomers were chosen to minimize the effects of the base identity on the CID spectra. A full complement of ions representing the w, y, b, and d series27 and their respective dehydration products were observed (Supporting Information, Figure S1), although it is noted that, in homopolymers with a 3′-terminal phosphate, the d and y ions are isobars. The single most striking feature is the complete absence of any a - base (a - B) or base (uracil-) fragment ions in the mass spectrum of Ψ4p, which are otherwise quite common in all oligonucleotides.28 This finding reflects the resistance of the Ψ C-C glycosidic bond to cleavage29 and suggests that b and d series ions are not necessarily products of the original a - B backbone scission event, as has been proposed in the case of conventional oligonucleotides.30,31 The other outstanding feature of the spectrum is the observation of pseudouridinecharacteristic ions in the region of the spectrum below m/z 250 (Figure 1). Proposed structures for these ions are schematically depicted in Figure 2. These represent the nucleoside anion (m/z 243), its multiple dehydration products (m/z 225, 207, 189), and products of fragmentation through the sugar moiety (m/z 165, 153, 139). Ions are also observed that represent multiple fragmentation events (m/z 164, 122, 110). In addition, a minor but Ψ-specific ion of m/z 139 is often observed in product ion mass spectra of many Ψ-containing oligonucleotides. Its structure assignment is uncertain and is under investigation. Several experiments were conducted in order to ascertain the origins of the pseudouridine fragments, including precursor ion scans for many of the fragments, as well as constant neutral loss scans for losses of 43 and (27) McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70. (28) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2004, 237, 197-241. (29) Rice, J. M.; Dudek, G. O. Biochem. Biophys. Res. Commun. 1969, 35, 383388. (30) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Chem. Soc. 1993, 115, 1208512095. (31) 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.

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Figure 1. Low m/z region of the product ion mass spectrum of Ψ4p, showing a number of ions characteristic of the presence of pseudouridine.

Figure 2. Schematic representations of fragment ions arising from dissociation of oligonucleotides containing Ψ. RDA, retro-Diels-Alder reaction. Decomposition pathways are inferred in part from precursor ion scan measurements. Sites of deprotonation are not known, and all structures are drawn as neutral species.

60 u. Representative mass spectra reflecting these inherently complex pathways28 are shown in Supporting Information (Figures S2-S4). Dehydration Product Ions. In addition to the aforementioned b, d, y, and w ions and their respective dehydration products, ions corresponding to doubly dehydrated products are also observed, e.g., b2 - 2H2O- (m/z 513) and d/y2 - 2H2O- (m/z 593). These multiple losses of water in Figure S1C are not limited to sequence series ions. The nucleotide di- and monophosphate anions (m/z 403 and 323, respectively) both exhibit loss of one (m/z 385, 305) and two molecules of H2O (m/z 367, 287). The monophosphate anion even shows loss of three waters at m/z 269. This

facile loss of water from the molecular ion and various fragment ions is a hallmark of pseudouridine mass spectra, observed even in the absence of collisional activation, by EI,32 CI,33 FAB,34,35 and thermospray17 and electrospray10 ionization methods. Product ions in Figure 2 are observed that correspond in mass to the loss of one, two, and three molecules of water from the intact nucleoside. The doubly dehydrated ion at m/z 207 is generally the most abundant of the characteristic small fragment ions. Interestingly, and in contrast to expectations from the positive ion EI mass spectrum in which pathways supported by metastable ions were reported,29 this ion does not seem to be derived from the m/z 243 and 225 ions as expected. Rather it seems to have its origin primarily from the dehydrated monophosphate at m/z 305 and, to a lesser extent, from members of the principal sequence series ions (Figure S4E). Furthermore, as measured using precursor ion scans, the nucleoside anion precursor, or at least the m/z 243 ion, derived from nozzle-skimmer dissociation of the Ψ4p tetramer, loses only a single molecule of water to yield the m/z 225 ion, and no further dehydrations are observed. Ribose Cleavage Product Ions. Ions are observed that correspond in mass to classical fragmentation pathways for nucleoside collision-induced dissociation products. These fragments consist of the intact base with portions of the sugar moiety.36 They have been reported previously in both positive and negative ion spectra and are generally independent of the ionization method, being observed in EI,32,37 CI,38 and FAB35 mass spectra. Included in this category is m/z 153 (B + 42), the base plus C-1′, C-2′, and O-2′. Absent any contradictory evidence, there is no compelling reason to assume these ions are anything other than their counterparts seen using other forms of ionization and activation. As with the m/z 207 ion, both m/z 139 and 153 appear to be generated from molecular species and from fragments formed via principal cleavages through the backbone (b, d, y, w, ions) with the m/z 139 ion favoring the direct route from those precursors somewhat more than the m/z 153 ion, which tended to favor smaller, single-residue (mononucleotide) fragments. In both cases, Na salt adducts seem to stabilize the transition state, as there seems to be a relatively significant population of these ions in the precursor scan. These ions include Ψ>p + Na (m/z 327) as precursor for m/z 139 and Ψ>p + Na (m/z 345) and Ψ + Na (m/z 265) for m/z 153. Base Cleavage Product Ions. Essentially all uracil-containing ions smaller than a mononucleotide undergo characteristic dissociation in the pyrimidine moiety analogous to the retro-DielsAlder (RDA) reaction,29 losing either NCO or HNCO, e.g., m/z 207 f 164 in Figure 1. In a study of the mass spectra of protonated (32) Smith, D. L.; Schram, K. H.; McCloskey, J. A. Biomed. Mass Spectrom. 1983, 10, 269-275. (33) Wilson, M. S.; McCloskey, J. A. J. Am. Chem. Soc. 1975, 97, 34363444. (34) Crow, F. W.; Tomer, K. B.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E. Anal. Biochem. 1984, 139, 243-262. (35) Slowikowski, D. L.; Schram, K. H. Nucleosides Nucleotides 1985, 4, 347376. (36) McCloskey, J. A. Acc. Chem. Res. 1991, 24, 81-88. (37) McCloskey, J. A. In Basic Principles in Nucleic Acid Chemistry; Ts’o, P. O. P., Ed.; Academic Press: New York, 1974; Vol. 1, pp 209-309. (38) Slowikowski, D. L.; Schram, K. H. Nucleosides Nucleotides 1985, 4, 309345.

uracil and its isotopically labeled derivatives, Nelson39 found that nearly 87% of the loss could be accounted for by N-3, C-2, and O-2, but we are unaware of any corresponding study of deprotonated uracil. The effects of substitution at N-1 and N-3 should be established a priori before informed interpretation of the CID mass spectra of oligonucleotides containing the naturally occurring nucleosides40 m1Ψ, m3Ψ, and m1acp3Ψ is attempted. There is probably little effect noticeable on this pathway due to 2′-Omethylation (Ψm). Special Phosphorylated Product Ions. A fragment ion corresponding in mass to the nucleoside triphosphate, m/z 483, and of probable structure ppΨp, is observed in the spectrum of Ψ4p, but the analogous ion is not observed in either of the control sequences (Figure S1). Although similar ions resulting from a phosphate rearrangement, i.e., ppN, have been reported previously,41 this is the first observation of the triphosphate analogue. In DNA, where p and f (f represents the substituted furan at the 3′ terminus of an a - B ion31) are nominal isobars, the ppN ion is indistinguishable from its structural isomers, pNp and Npf, both of which require multiple backbone scission events, except for the 5′ terminal position. The occurrence of both pNp (or ppN) and Npf has been confirmed by exact mass measurement41 and isotopic labeling experiments.42 Those same experiments excluded the existence of the ppNp ion and permitted only the pNpf structure. Sequence Placement of the Pseudouridine. Experiments were conducted in order to ascertain the existence of spectral features that would permit the sequence assignment of the Ψ residue. Initial research was focused on the pair of isomers UUUUUUUUA and UUUUΨUUUA. These sequences were chosen to minimize the effects of base composition on the product ion spectra and to distinguish between the d/w ions and the b/y ions, which are isobaric for homopolymers. The A residue was positioned at the 3′ terminus as an end marker; loss of the base from this position is disfavored. The precursor ion was the (M 4H)4- ion at m/z 678.1, (collision energy 50 eV; collision gas thickness 300 × 10-15 atoms/cm2 (∼6 mTorr)). The mass spectra (Sciex instrument) are shown in Figure 3. Complete sequence representations for the principal ion series are observed; for clarity they are not indicated in the figure. As the nature of the modification is mass-silent, there are no shifts in m/z values that would provide a clear indication of the position of modification, thus accounting for the high degree of similarity between the spectra and requiring a more detailed analysis. Portions of the spectra containing the ions associated with cleavage on the 3′ side of the site of modification are presented in a detail magnification in Figure 3C and D. The relative abundance of the w42- ion is elevated by a factor of 9.7 in the spectrum of the Ψ-containing oligonucleotide versus the control. The w41- ion abundance was similarly increased by a factor of 9.0 (data not shown), while the a52- ion exhibited a 396% increase in (39) Nelson, C. C.; McCloskey, J. A. J. Am. Soc. Mass Spectrom. 1994, 4, 339349. (40) Motorin, Y.; Grosjean, H. In Modification and Editing of RNA; Grosjean, H., Benne, R., Eds.; ASM Press: Washington, DC, 1998; pp 543-549. (41) Hettich, R. L.; Stemmler, E. A. Rapid Commun. Mass Spectrom. 1996, 10, 321-327. (42) Pomerantz, S. C.; McCloskey, J. A., unpublished experiments, 1995.

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Figure 3. Product ion mass spectra of the isomeric oligonucleotides (A) UUUUUUUUA and (B) UUUUΨUUUA. Panels C and D show magnified sections of spectra from (A) and (B), illustrating enhanced abundances of a5 and w4 ions due to presence of Ψ.

response. The singly charged a5 ion was outside the scan range of the experiment, and neither the a5 nor w5 ions were observed in the triply charged state. An alternative and compelling view of the effect is found in Figure 4, where the product ion relative abundances have been summed over the observed charge states and subsequently normalized to pertinent members of their respective series. The basis for normalization of the w series is the w1 ion, previously identified as usually being the most abundant of the w series43 or, more generally, any ion bearing the 3′ terminus. Since no corresponding correlation has been deduced or formalized for relative ion abundance where the charge is retained on the 5′ terminus, the basis for normalization for the a series was chosen to be the a2 ion. This ion was selected as there was no ion abundance observed for the a1 species in the mass spectrum of the control isomer. Either of these criteria for normalization reflects, to varying degrees, the experimental conditions, e.g., precursor charge state and abundance, collision energy, and collision gas pressure. Although the data represented in this figure indicate that both of the isomers generally behave in a similar fashion with respect to their dissociation pathways, there are clear variances observed at the positions around the modification site. The apparently abundant w5 ion in both the control and modified isomers in the Figure 4B graph is ascribed (43) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal. Chem. 1996, 68, 1989-1999.

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Figure 4. Normalized sum, observed over all charge states, of product ion abundances for two oligonucelotide isomers. (A) a ion series, normalized to the a2 ion. (B) w ion series, normalized to the w1 ion. Vertical arrows represent abundance differences at the site of pseudouridylation.

to isobaric interference from the isotope profile of the w2 - H2O - Ura fragment. Inspection of these graphs indicates that the dissociation products and pathways from these isomers are generally the same, as there is very little scatter in the individual data points. The sole exception are the ions resulting from the backbone cleavage between the C-3′ and O-3′ of the Ψ residue, resulting in the a5 and w4 cleavage-complementary ions, which are both significantly elevated over the control sequence. Several groups have posited the existence of the a series ions, both in ESI44 and in MALDI;45 however, it has perhaps not been recognized that the proposed a ions result in a structure that is isobaric with the b - H2O ion, which is often observed. We postulate that these ions are enhanced due to the inability to lose the Ψ base (which would otherwise constitute a major pathway leading to dissociation at this point in the chain28,30), a consequence of the increased C-C glycosidic bond strength of Ψ.29 The increased abundance of these ions arises from the failure to form the secondary product of the collision, i.e., the base, either as an (44) Bartlett, M. L.; McCloskey, J. A.; Manalili, S.; Griffey, R. H. J. Mass Spectrom. 1996, 31, 1277-1283. (45) Wang, B. H.; Hopkins, C. E.; Belenky, A. B.; Cohen, A. S. Int. J. Mass Spectrom. Ion Processes 1997, 169/170, 331-350.

Table 1. Selected Ion Dissociation Pathways for Detection of Pseudouridine-Containing RNA Oligonucleotides

a

MRM pathwaya

principal selectivity

m/z 207 f 164 m/z 225 f 165 m/z 668 f 207

detection of Ψ at nonterminal oligonucleotide positions presence of Ψ at 5′-terminal oligonucleotide position occurrence of ΨpGp from RNase T1 hydrolysis of the RNA sequence ...GpΨpGp...

Negative ions; see Figure 1.

anion or as a neutral. Thus, all of the energy of the collision at this point in the chain is channeled into only two principal products, the complementary a and w ions, rather than three or more. Sequence Context Considerations. Further investigations focused on the sequence GCUUAGUCA, and the monosubstituted Ψ isomers at each of the U positions (GCΨUAGUCA, denoted 9R; GCUΨAGUCA, 9β; and GCUUAGΨCA, 9γ). This sequence was randomly generated from a number of candidate 8-10-mers and was selected on the basis of minimized isobaric interferences predicted by computer-simulated CID spectra (software development by the author). Product ion spectra were acquired from the (M - 4H)4- precursor (m/z 702.7) for each of the individual isomers. Complete sequence representations for all principal series ions could be assigned for each of the isomers. Examination of the pertinent ions reveals the previously discussed enhancement of cleavage-complementary ions on the 3′ side of the site of modification. To compare enhancements from different sequences and experimental conditions in a more facile manner, an independent measure of the enhancement is defined in eq 1. EF is

EF )

(Σ wn,Ψ)/w1,Ψ (Σ wn,U)/w1,U

Ψ is to the center of an oligonucleotide, the less likely is backbone scission44 that vitiates a more dramatic enhancement from Ψ presence at those positions.

(1)

the enhancement factor, the numerator is equal to the ratio of the wn ion intensity, summed over all observed charge states, to the abundance of the w1 ion in a Ψ-containing sequence, and the denominator is the same ratio for the control (U instead of Ψ) sequence. A similar expression is defined for the a series ions; however, the denominator of each ratio is defined as the intensity of the a2 ion, the first well-characterized member of this series. When no enhancement is observed, the EF is equal to unity. Plots of the EF versus position for the three pseudouridylated oligonucleotides are shown in Figure 5. The EF values all cluster about 1.0 with relatively little scatter, except for the complementary ions resulting from scission of the backbone between the C-3′ and O-3′ at a Ψ position. The w9 ion is not included in the plot, as it is equivalent to the molecular ion precursor. The termini ions in the a series plot are not well-characterized due to their low abundance and are not included in the plot. In these three isomers, the a ions exhibit enhancement factors ranging from about 3 to 4.5, while the w series demonstrate EF values of approximately 3.5-8. There is not a significant enough difference in either of these series of enhancement factors to draw a single conclusion about the influence of the local sequence context, and the extent to which neighboring base identity plays a role. With respect to the w series ions, there is less consistency among the data, although the principal effects of Ψ location are evident. The closer

Figure 5. Enhancement factors as a function of Ψ-position for three monosubstituted isomeric oligonucleotides, showing the effects of pseudouridylation. (A) a ion enhancement factor. (B) w ion enhancement factor.

Use of Selective Dissociation Pathways for Detection and Sequence Placement of Ψ in Oligonucleotide Mixtures. Preliminary Development of the Assay. Results described in the preceding sections on the characteristic fragmentation pathways of Ψ-containing oligonucleotides allowed the design of selective ion dissociation pathways for detection of pseudouridine using MS/MS and MRM. By direct combination of mass spectrometry with liquid chromatography, the method can then in principle be applied to mixtures of oligonucleotides, as for instance resulting from nuclease digests of RNA. Three useful reaction pathways are summarized in Table 1, and examples of their applications are given in the following sections. Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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The principal MRM analysis selected for the assay was the reaction from m/z 207 to 164. This transition was selected on the basis of the following criteria: (1) the m/z 207 ion is the most abundant of the characteristic Ψ fragment ions, thus generating the largest population of precursor ions, and (2) the further retroDiels-Alder decomposition of the m/z 207 ion would diminish nonspecific signals arising from minor alternate fragmentation pathways to ion m/z 164. Generation of the m/z 207 ion via nozzle-skimmer dissociation at the ion source, followed by its subsequent mass selection with Q1, dissociation in the q2 gas collision cell, and detection of the m/z 164 product in Q3 was found to be an efficient route for the desired assay. Experimental conditions for the assay were originally established empirically by infusion of UUUUΨUUUA and monitoring of the desired products, m/z 207 (Q1) and 164 (Q3), in real time via the data system tune page display. This procedure established ∼75 V as the nozzle-skimmer potential difference (with the Z-spray ion source) and ∼14 eV as the collision energy with an argon pressure of ∼3 × 10-3 mbar in the collision cell. Confirmation of the assay conditions was carried out by HPLC introduction of this compound and each of the pseudouridylated 9-mers and the control 9-mer discussed previously (data not shown). tRNA: A Small Natural RNA Model System. Initial experiments to determine the feasibility of the overall approach were conducted on E. coli tRNA2Tyr (88 nt; Mr ∼28 000). This system was selected because (1) the RNA sequence, including modifications, is known,46 (2) the number of oligonucleotides generated by RNase T1 digestion is easily experimentally manageable, and (3) there are two Ψs present in nuclease product oligonucleotides, having differing size and sequence environments. Assay of the digest for the presence of Ψ-containing oligonucleotides was performed on a 20-pmol scale. The result of the screening LC/ MS run is shown in Figure 6, which clearly indicates the presence of two Ψ-containing oligonucleotides. Assignment of the predicted RNase T1 fragments to specific HPLC peaks, as indicated in Figure 6A, were predicated solely upon experimentally determined Mr values and the reported tRNA sequence:46 Mr 1294.8, component 8 and Mr 4100.7, component 13 in Figure 6A. Although there are a few other minor responses in the MRM chromatogram, these are judged to arise from contaminating tRNA isoacceptors, which are evident in the HPLC UV trace, and no attempt was made to identify the minor constituents. Sequence determinations on the two designated Ψ-containing digestion products were carried out by LC/MS/MS using a Q-Tof 2 instrument. The product ion CID spectrum, acquired following mass selection of the (M - 3H)3- precursor ion, for the tetramer m5UΨCGp is shown in Figure 7A. The presence of Ψ in the molecule is confirmed by the characteristic fragment ions found in the low m/z region of the spectrum (e.g., m/z 225, 207, 164, 139), while placement of the Ψ is readily accomplished by examination of the appropriate sequence series ions. The w series ions are represented through w3,, verifying the basic sequence of the tetramer and placing m5U at the 5′ terminus. The key element in assigning the position of the Ψ residue is the very abundant w2 ion, which is ∼10% more abundant than the w1 ion at the 1charge state but an extraordinary 10-fold more abundant at the 2- charge state. The w3 ion is present at reasonable and customary (46) Sprinzl, M.; Vassilenko, K. S. Nucleic Acids Res. 2005, 33, D139-D140.

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Figure 6. Selective detection of pseudouridylated oligonucleotides by LC/MS analysis using MRM, of aN RNase T1 digest of E. coli tRNA2Tyr. (A) HPLC separation with UV detection. (B) MRM recording from the reaction pathway m/z 207 f 164, showing presence of internal Ψ in two of the oligonucleotide products.

Figure 7. Sequence mass spectra showing sites of Ψ in the pseudouridylated oligonucleotides detected in Figure 6B. (A) Sequence mass spectrum of component 8 in Figure 5A. Asterisk, (M 3H)3- precursor ion. Not annotated, m/z 139. (B) Sequence mass spectrum of component 13 in Figure 5A. Asterisk, (M - 7H)7precursor ion. Ψ fingerprint ions not annotated are m/z 122, 139, 153, and 189.

abundance levels. The only a - B series ion present is the lowabundance a3 - Cyt, detected at the 1- and 2- charge states (m/z 739.5, 369.2; not annotated). This is expected as the a1 - B ion is undefined in terms of sequence, a2 - Ura does not occur when the glycosidic bond is C-C (Ψ), and a4 - Gua is unlikely as loss of the 3′ terminal base is disfavored. The sequence placement is further corroborated by the very substantial y2 and a2 ions. The

y2 ion is presumed to derive from the w2 ion by loss of phosphate,31 while the a series ion is usually not observed in such plenitude, except when it is at a Ψ residue. The Ψ therefore occurs at tRNA position 55, a highly conserved modification site in tRNA.46 The product ion mass spectrum of the oligonucleotide 12-mer containing the tRNA anticodon is shown in Figure 7B. This spectrum is a powerful example of the strength of mass spectrometry for the primary structure determination of small, modified oligonucleotides. Even in the presence of the two hypermodified residues, queuosine (Q) and 2-methylthio-N6-isopentenyladenosine (ms2i6A), essentially complete series representations for the w and a - B series were identified (not annotated in Figure 7B; summarized in Table S1 in Supporting Information). The placement of the Ψ at position 9 is unequivocal, supported by the very robust w32- ion, the second most intense peak in the mass spectrum, and four times the amplitude of the w1 ion, which is otherwise generally the most abundant of the w series ions. Although ion a9 is not abundant, further support for this placement in the sequence is the corresponding notable abundance of ion y3 compared with ions y2 and y4. There is no evidence for Ψ at either of the U positions 3 or 5, and the positioning of Ψ at position 10 (corresponding to tRNA position 39 as required by the published sequence46) is unambiguous. E. coli 16S rRNA: A Large Model RNA System. There is only one Ψ in the 1542 residues of E. coli 16S rRNA (Mr ∼495 000); it is located at position 516.47 The corresponding gene sequence predicts the local RNA sequence prior to posttranscriptional modification to be ...515-GUG-517.... If RNase T1 had been utilized, the resulting ΨGp dinucleotide could not readily be placed at any of 32 possible ...GUG... sites in the molecule, a common circumstance when dealing with large RNAs. To get a longer sequence, which could be uniquely placed, ribonuclease U2, which is relatively specific for A, was employed. The enzyme preferentially cuts at most purine residues (A and G), and leaves a 2′,3′cyclic phosphate moiety at the 3′ terminus of the resultant oligonucleotide (...A>p or ...G>p). Preliminary examination of the Mr and MRM data (i.e., from two sequential experiments) revealed multiple responses for the presence of pseudouridine (data not shown). The multiply charged molecular species that tracked the MRM response with the greatest fidelity were deconvoluted to Mr values of 2218.3 and 3159.3 (components B and C in Figure 8A). Analysis of the product ion mass spectrum of component B (Figure 8B) did not reveal the presence of any w series ions in apparent excess abundance. There are 24 7-mers predicted by the gene sequence, which do not contain an A (the adenine base was not observed in the CID spectrum), of which 13 terminate in ...G>p. Since the product ion spectrum (presented using the MaxEnt3 deconvolution format in Figure 8B) did not reveal any extraordinary ion abundances for putative w series ions, the presumed sequence was one of the two that ended in ...UG>p, where the enhancement effect of pseudouridylation would be masked as the normally abundant w1 ion. Manual interpretation of the CID spectrum best supported the sequence 511CUCCGΨG>p-517, for which a nearly complete set of wn (n ) 1, 2, 4, 5) and an - B (n ) 2-5) ions could be found. A very low (47) Bakin, A.; Kowalak, J. A.; McCloskey, J. A.; Ofengand, J. Nucleic Acids Res. 1994, 22, 3681-3684.

Figure 8. Sequence placement by LC/MS/MS of the single Ψ residue in E. coli 16S ribosomal RNA, from direct analysis of an RNase U2 digest. (A) HPLC separation of products; UV detection at 260 nm. (B) and (C) mark the elution points of the oligonucleotides whose mass spectra are shown in panels B and C, respectively. (B) sequence mass spectrum of component B in panel A, from the (M 4H)4- (m/z 553.31) precursor ion, 60 eV collision energy (Elab); represented in MaxEnt3 format. Peaks marked by asterisks are harmonic artifacts of the deconvolution algorithm. (C) Sequence mass spectrum of component C in panel A, from the (M - 6H)6- (m/z 525.06) precursor ion; 90 eV collision energy (Elab), MaxEnt3 format.

abundance w3 - H2O ion was observed, and this assignment was corroborated by the presence of yn (n ) 1-5) series ions, with y3 also poorly represented in the spectrum. The relatively abundant ion at m/z 1791.3 could then be assigned as the a6 ion, the cleavage complement of the w1 ion, implying the presence of Ψ at RNA position 516, the penultimate residue in this oligonucleotide. A similar approach was employed for the other principal oligonucleotide that elicited an MRM response (Mr 3159.3). There are six sequences predicted at this mass, only one of which terminates in ...A>p, and none terminate in ...G>p. The sequence that ended in ...A>p corresponds to a three-residue extension (511CUCCGΨGCCA>p) of the oligonucleotide previously discussed above and was indeed the expected sequence for the ribonuclease U2 fragment. Issues of data interpretation unique to sequences ending in the ...A>p motif will be detailed in a subsequent report (Guymon et al., manuscript in preparation). When an A>p-3′ precursor molecular ion is selected that has 50% or fewer of the phosphates charged, the only product ion of significant abundance is the loss of neutral adenine, even though loss of the base from the 3′ terminus is generally disfavored.30 This phenomenon is also observed, albeit to a lesser extent, in the MaxEnt3 deconvolution of the product ion spectra derived from the (M - 6H)6- precursor of the oligonucleotide 10-mer (Figure 8C). All of the w series ions are small or nonexistent (w3-w5). For the predominant series, Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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which is representative of the sequence from the 3′ terminus is the w - Ade series, which is found in 2-5-fold greater abundance than the w series, some members of this series are found even in the complete absence of the corresponding w series ion. The presence of elevated responses for the w4 - Ade, y4, and y4 Ade, in concert with a robust a6 ion, is sufficient to uniquely place the Ψ residue at position 6 of the 10-mer. These data further confirm the presence of Ψ specifically at position 516 in the 16S rRNA.47 Refinement of the Assay. During systematic application of the assay to other 16S rRNAs (to be reported elsewhere), it was noted that the m/z 207 f 164 MRM response had ∼10-20-fold poorer sensitivity when the Ψ residue was located at the 5′ terminus of an oligonucleotide (see Figure S5). A series of model compounds synthesized for other studies was used to investigate these effects. The control sequence was UAACUAUAACG and each of the three monopseudouridylated isomers. Examination of the data (not shown) for ΨAACUAUAACG revealed a markedly distinct dissociation behavior, which had earlier been masked by the presence of the other Ψ residues in the original Ψ4p analysis. The preferred fragmentation pathway for a 5′ terminal Ψ residue is formation of the a1 ion at m/z 225, followed by loss of 60 u to yield the m/z 165 ion (see Figure 1). In this special case, these two ions are present in much greater yield than all of the other characteristic fragment ions. Since this sequence motif was being observed with some regularity in the analysis of RNase T1 digests of other RNAs, the assay was modified to incorporate the MRM transition of m/z 225 f 165 for selective detection of 5′-terminal Ψ (Table 1). This MRM transition did not have good sensitivity, however, for the ΨGp dinucleotide (arising from the sequence ...GΨG..., often found in ribosomal RNAs), as the m/z 225 ion (a1) could not favorably compete for retention of charge with the cleavage-complementary pGp ion (w1) formed when the dinucleotide dissociates. The assay was thus further modified to include the MRM transition m/z 668 f 207, which represents the dissociation of the singly charged ΨGp dinucleotide to the doubly dehydrated nucleoside. The doubly charged dinucleotide could not be selected as the precursor due to interference from the solvent background. Procedurally, this MRM transition is monitored only at the beginning of the HPLC elution profile, until all of the dinucleotides elute. Subsequently, the transitions indicative of both internal and 5′-terminal Ψ are examined (in the same run), in alternating fashion with the molecular mass scan function. The full assay is illustrated in Figure 9 for the RNase T1 digest of E. coli 23S rRNA (2904 nt; Mr ∼932 000). The single strong response at 45.5 min in Figure 9C is due to the Ψ at position 1911, with only very minor contributions from Ψ1917. The extent to which the minor dissociation pathway (m/z 225 f 165) for internal Ψ occurs may be estimated from the other responses that arise from oligonucleotides that only contain internal Ψ. In the absence of Ψ1917, the response in Figure 8D at 45.5 min would be ∼15-fold lower, if it were only due to Ψ1911. Overall, the data in Figure 9B-D account for the nine Ψ (in addition to m3Ψ-1915) reported in the E. coli large subunit rRNA.48, 49 (48) Ofengand, J.; Fournier, M. J. In Modification and Editing of RNA; Grosjean, H., Benne, R., Eds.; ASM Press: Washington, DC, 1998; pp 229-253. (49) Ofengand, J. FEBS Lett. 2002, 514, 17-25.

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Figure 9. Modified LC/MS/MS assay for presence of various pseudouridylated oligonucleotides in a RNase T1 digest of E. coli 23S ribosomal RNA. (A) HPLC separation of products; UV detection at 260 nm. (B) MRM response for ΨGp (m/z 668 f 207). (C) MRM response for presence of 5′-terminal Ψ (m/z 225 f 165). (D) MRM response for internal Ψ (m/z 207 f 164).

CONCLUSIONS Two mass spectrometry-based approaches have been developed to address the vexing problem of detection and sequence placement of pseudouridine in small (n e 15) oligoribonucleotides, in particular those resulting from selective RNase digestion of large RNAs such as those present in the ribosome. In the first method, Ψ can be reliably detected from the presence of fragment ions characteristic of the C-C glycosidic bond, which are generated in lieu of major pathways otherwise associated with favorable cleavage of the normal C-N glycosidic bond. In the second method, sequence placement is indicated by enhanced abundance of a- and w- or y-type ions at the site of pseudouridylation and by absence of the a - B ion type due to resistance to glycosidic bond cleavage in C-nucleosides. Although in the model studies presented here, the sequence positions of pseudouridine are clearly marked by abundance changes, it is likely that given the potentially large number of sequence variations, the effects may be masked by unusual or unknown sequence context effects. Unless the assignments are obviously straightforward, unmodified control sequences should be examined for comparison if feasible. Both methods can be applied using direct sample flow into the electrospray ion source, but substantial advantages accrue when applied using LC/MS, permitting analysis of complex oligonucleotide mixtures. In particular, three MRM scan functions can be efficiently applied using LC/MS in which selective detection of Ψ can be effected for residues in the inner oligonucleotide chain, at the 5′ terminus, and in the common dinucleotide U/ΨpGp.

There are two principal limitations of the method. The first arises in very complex RNA digests in which the coelution of oligonucleotides confounds the time alignment of Ψ-related MS signals with molecular mass-defining responses. The second is difficulty in dealing with substoichiometric levels of Ψ at any given position, resulting in insufficient S/N for definitive sequence analysis. However, the method is complementary to the reverse transcriptase/gel-based method of Ofengand,19 in which sites of Ψ placement in the overall RNA structure can be independently derived for matching against the data from the MS method.

ACKNOWLEDGMENT This work was supported by Grant GM29812 from the National Institutes of Health. We are grateful to R. Guymon and P. F. Crain for assistance in assembling the manuscript. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 21, 2005. Accepted May 27, 2005. AC058023P

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