Collision-Induced Dissociation of Intact Duplex and Single-Stranded

Oct 23, 2008 - The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied usin...
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Anal. Chem. 2008, 80, 8501–8508

Collision-Induced Dissociation of Intact Duplex and Single-Stranded siRNA Anions Teng-yi Huang, Jian Liu, Xiaorong Liang, Brittany D. M. Hodges, and Scott A. McLuckey* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084 A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies. RNA interference (RNAi) is a phenomenon whereby gene expression is inhibited post-transcriptionally through mRNA degradation, chromatin modification, and translational arrest in eukaryotic cells. Noncoding RNAs of 21-25 nucleotides, such as small interfering RNA (siRNA) and micro-RNA (miRNA) are the effectors of RNAi pathway. These small noncoding RNAs are enzymatically processed from endogenous long double-stranded RNAs and pre-micro-RNAs and are incorporated into RNA-induced silencing complexes (RISCs) to effect RNA interference.1 Recently, the abnormal expression of small noncoding RNAs has been correlated to a wide spectrum of human diseases. For example, various miRNAs are found to be up- or down-regulated * To whom correspondence should be addressed. Phone: 765-494-5270. Fax: 765-494-0239. E-mail: [email protected]. (1) Dorsett, Y.; Tuschl, T. Nat. Rev. Drug Discovery. 2004, 3, 318–29. 10.1021/ac801331h CCC: $40.75  2008 American Chemical Society Published on Web 10/24/2008

in different types of cancers. These cancer-related miRNAs were found to target oncogenes or tumor suppressor genes.2 A study on Parkinson’s disease has identified an miRNA as a regulator of the maturation and function of midbrain dopaminergic neurons.3 The important roles played by various noncoding RNAs have led to significant efforts in the study of RNomics, and new classes of noncoding small RNA have been discovered and functionally characterized.4-8 Their applications as therapeutics have also been explored.1,9-11 The most commonly used methods for identification of noncoding RNAs are through cloning and sequencing; new methods such as microarray and reverse transcription polymerase (2) Calin, G. A.; Croce, C. M. Cancer Res. 2006, 66, 7390–7394. (3) Kim, J.; Inoue, K.; Ishii, J.; Vanti, W. B.; Voronov, S. V.; Murchison, E.; Hannon, G.; Abeliovich, A. Science 2007, 317, 1220–1224. (4) Mattick, J. S. EMBO Rep. 2001, 2, 986–991. (5) Mattick, J. S. Science 2005, 309, 1527–1528. (6) Carninci, P.; Kasukawa, T.; Katayama, S.; Gough, J.; Frith, M. C.; Maeda, N.; Oyama, R.; Ravasi, T.; Lenhard, B.; Wells, C.; Kodzius, R.; Shimokawa, K.; Bajic, V. B.; Brenner, S. E.; Batalov, S.; Forrest, S. R. R.; Zavolan, M.; Davis, M. J.; Wilming, L. G.; Aidinis, V.; Allen, J. E.; Ambesi-Impiombato, X.; Apweiler, R.; Aturaliya, R. N.; Bailey, T. L.; Bansal, M.; Baxter, L.; Beisel, K. W.; Bersano, T.; Bono, H.; Chalk, A. M.; Chiu, K. P.; Choudhary, V.; Christoffels, A.; Clutterbuck, D. R.; Crowe, M. L.; Dalla, E.; Dalrymple, B. P.; de Bono, B.; Della Gatta, G.; di Bernardo, D.; Down, T.; Engstrom, P.; Fagiolini, M.; Faulkner, G.; Fletcher, C. F.; Fukushima, T.; Furuno, M.; Futaki, S.; Gariboldi, M.; Georgii-Hemming, R.; Gingeras, T. R.; Gojobori, T.; Green, R. E.; Gustincich, S.; Harbers, M.; Hayashi, Y.; Hensch, T. K.; Hirokawa, N.; Hill, D.; Huminiecki, L.; Iacono, M.; Ikeo, K.; Iwama, A.; Ishikawa, T.; Jakt, M.; Kanapin, A.; Katoh, M.; Kawasawa, Y.; Kelso, J.; Kitamura, H.; Kitano, H.; Kollias, G.; Krishnan, S. P. T.; Kruger, A.; Kummerfeld, S. K.; Kurochkin, I. V.; Lareau, L. F.; Lazarevic, D.; Lipovich, L.; Liu, J.; Liuni, S.; McWilliam, S.; Babu, M. M.; Madera, M.; Marchionni, L.; Matsuda, H.; Matsuzawa, S.; Miki, H.; Mignone, F.; Miyake, S.; Morris, K.; Mottagui-Tabar, S.; Mulder, N.; Nakano, N.; Nakauchi, H.; Ng, P.; Nilsson, R.; Nishiguchi, S.; Nishikawa, S.; Nori, F.; O’Hara, O.; Okazaki, Y.; Orlando, V.; Pang, K. C.; Pavan, W. J.; Pavesi, G.; Pesole, G.; Petrovsky, N.; Piazza, S.; Reed, J.; Reid, J. F.; Ring, B. Z.; Ringwald, M.; Rost, B.; Ruan, Y.; Salzberg, S. L.; Sandelin, A.; Schnieder, C.; Schonbach, C.; Sekiguchi, K.; Semple, C. A. M.; Seno, S.; Sessa, L.; Sheng, Y.; Shibata, Y.; Shimada, H.; Shimada, K.; Silva, D.; Sinclair, B.; Sperling, S.; Stupka, E.; Sugiura, K.; Sultana, R.; Takenaka, Y.; Taki, K.; Tammoja, K.; Tan, S. L.; Tang, S.; Taylor, M. S.; Tegner, J.; Teichmann, S. A.; Ueda, H. R.; van Nimwegen, E.; Verardo, R.; Wei, C. L.; Yagi, K.; Yamanishi, H.; Zabarovsky, E.; Zhu, S.; Zimmer, A.; Hide, W.; Bult, C.; Grimmond, S. M.; Teasdale, R. D.; Liu, E. T.; Brusic, V.; Quackenbush, J.; Wahlestedt, C.; Mattick, J. S.; Hume, D. A.; Kai, C.; Sasaki, D.; Tomaru, Y.; Fukuda, S.; Kanamori-Katayama, M.; Suzuki, M.; Aoki, J.; Arakawa, T.; Iida, J.; Imamura, K.; Itoh, M.; Kato, T.; Kawaji, H.; Kawagashira, N.; Kawashima, T.; Kojima, M.; Kondo, S.; Konno, H.; Nakano, K.; Ninomiya, N.; Nishio, T.; Okada, M.; Plessy, C.; Shibata, K.; Shiraki, T.; Suzuki, S.; Tagami, M.; Waki, K.; Watahiki, A.; Okamura-Oho, Y.; Suzuki, H.; Kawai, J.; Hayashizaki, Y. Science 2005, 309, 1559–1563. (7) Mehler, M. F.; Mattick, J. S. J. Physiol. 2006, 575, 333–341. (8) Aravin, A. A.; Hannon, G. J.; Brennecke, J. Science 2007, 318, 761–764. (9) Ku, G.; McManus, M. Y. Hum. Gene Ther. 2008, 19, 17–26. (10) Chiu, Y. L.; Rana, T. M. RNA 2003, 9, 1034–1048. (11) Manoharan, M. Curr. Opin. Chem. Biol. 2004, 8, 570–579.

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chain reaction (RT-PCR) have also been demonstrated.12 On the basis of the unique structural characteristic of previously identified noncoding RNAs, bioinformatics methods have been developed to predict the candidates from the genomes of several organisms.13 Several studies have indicated that modified bases are closely related to the functions of noncoding RNAs;14-20 however, the aforementioned methods either are insensitive to the modifications, which are usually present in noncoding RNAs, or require the knowledge of the sequence. In addition, the potential of gene silencing via RNA interference has raised interest in the synthesis of siRNAs for therapeutic purposes. To efficiently effect RNA interference, base modifications or chemical analogues are usually introduced to the synthetic siRNA molecules in order to stabilize siRNA in vivo.1,9-11 Therefore, for the purpose of RNomics research and synthetic siRNAs characterization, it is desirable to develop a technique that is not only capable of providing primary sequence information but also sensitive in the identification of the naturally occurring modifications on the noncoding RNAs or the chemically modified nucleosides introduced in the synthetic siRNA molecules. Tandem mass spectrometry is a promising candidate for this application.21-23 Dissociation of RNA anions via collisional activation in the gas phase leads to RNA fragmentation through 5′ P-O backbone cleavage, which gives rise to the characteristic c/y-ion series.24-27 This cleavage mechanism competes with nucleobase loss and the subsequent backbone cleavage to yield (a-B)/w-ion series. The characteristic ions from both series can provide primary sequence information and allow the identification of the presence of modified nucleosides. The characterization of noncoding RNAs using tandem mass spectrometry has been demonstrated previously using a “bottom-up” approach.28-31 In a typical bottom-up experimental setup, the intact RNA is enzymatically digested into smaller oligomers and the digestion products are then subjected to mass (12) Hu ¨ ttenhofer, A.; Vogel, J. Nucleic Acids Res. 2006, 34, 635–646. (13) Washietl, S.; Hofacker, I. L.; Stadler, P. F. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2454–2459. (14) Grosjean, H.; Bjork, G.; Maden, B. E. H. Biochimie 1995, 77, 3–6. (15) Grosjean, H., Benne, R., Eds. Modification and Editing of RNA; ASM Press: Washington, DC, 1998. (16) Agris, P. F. Nucleic Acids Res. 2004, 32, 223–238. (17) Yu, B.; Yang, Z.; Li, J.; Minakhina, S.; Yang, M.; Padgett, R. W.; Steward, R.; Chen, X. Science 2005, 307, 932–935. (18) Blow, M. J.; Grocock, R. J.; van Dongen, S.; Enright, A. J.; Dicks, E.; Futreal, P. A.; Wooster, R.; Stratton, M. R. Genome Biol. 2006, 7, R27. (19) Helm, M. Nucleic Acids Res. 2006, 34, 721–733. (20) Alexandrov, A.; Chernyakov, I.; Gu, W.; Hiley, S. L.; Hughes, T. R.; Grayhack, E. J.; Phizicky, E. M. Mol. Cell 2006, 21, 87–96. (21) Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Anal. Chem. 1996, 68, 1989–1999. (22) Pomerantz, S. C.; McCloskey, J. A. Anal. Chem. 2005, 77, 4687–4697. (23) Thomas, B.; Akoulitchev, A. V. Trends Biochem. Sci. 2006, 31, 173–181. (24) Kirpekar, F.; Krogh, T. N. Rapid Commun. Mass Spectrom. 2001, 15, 8– 14. (25) Schu ¨ rch, S.; Bernal-Mendez, E.; Leumann, C. J. J. Am. Soc. Mass Spectrom. 2002, 13, 936–945. (26) Tromp, J. M.; Schu ¨ rch, S. J. Am. Soc. Mass Spectrom. 2005, 16, 1262– 1268. (27) Andersen, T. E.; Kirpekar, F.; Haselmann, K. F. J. Am. Soc. Mass Spectrom. 2006, 17, 1353–1368. (28) Ohara, T.; Sakaguchi, Y.; Suzuki, T.; Ueda, H.; Miyauchi, K.; Suzuki, T. Nat. Struct. Mol. Biol. 2007, 14, 349–350. (29) McCloskey, J. A.; Whitehill, A. B.; Rozenski, J.; Qiu, F.; Crain, P. F. Nucleosides Nucleotides 1999, 18, 1549–53. (30) Mengel-Jørgensen, J.; Kirpekar, F. Nucleic Acids Res. 2002, 30, e135. (31) Emmerechts, G.; Barbe´, S.; Herdewijin, P.; Anne´, J.; Rozenski, J. Nucleic Acids Res. 2007, 35, 3494–3503.

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spectrometry analysis. However, limitations, such as loss of molecular mass information for the intact molecules, congestion of peaks in a narrow mass range, and discrimination of certain oligomers from ionization of complex mixtures, could hamper its analytical utility. The enzymatic digestion of RNA may also introduce byproduct such as oligomers from incomplete digestion or 3′ cyclic phosphate. In addition, enzymatic digestion of a mixture of RNAs will lead to an even more complex mixture. Therefore, separation of the digestion mixture by liquid chromatography is usually needed before mass spectrometry analysis. In contrast to the “bottom-up” approach, a “top-down” approach, which does not involve enzymatic digestion of the analyte before mass spectrometry analysis, has been demonstrated and applied in proteomics research.32-34 In a typical top-down mass spectrometry experiment, intact biological macromolecules are first ionized by electrospray ionization (ESI), and then the ions of interest are isolated in the gas phase and subjected to dissociation. The molecular mass of the intact macromolecule and its characteristic fragment ions provide sufficient structural information for analyte identification. In this study, tandem mass spectrometry employing ion trap collisional activation and beam-type collisional activation has been applied to anions of a model intact siRNA duplex and its intact single strands. The dissociation phenomena of various charge states of duplex and single-stranded siRNAs were investigated under a range of excitation conditions. Ion trap collisional activation, which samples dissociation reactions at lower rates and, therefore, lower energies than beam-type collisional activation proved to be particularly useful for these ions. Complete sequencing of each strand was achieved by selective dissociation of the 5′ P-O bonds (c/y-ion series) or both the 5′ P-O bonds and the 3′ C-O bonds (a-B/w-ion series) through varying the activation conditions. Both the c/y- and a-B/w-ion series provide primary sequence information, (including the presence of post-transcriptional modification); however, the a-B/w-ion series can also be used to pinpoint the location (on the ribose or on the base) of the possible post-transcriptional modification on noncoding RNAs. The interpretation of the collision-induced dissociation (CID) product ion spectra was further facilitated by charge reduction of the multiply charged product ions to mostly singly and doubly charged ions via proton-transfer ion/ion reaction with benzoquinoline cations. The application of tandem mass spectrometry to intact strands of siRNA demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics. EXPERIMENTAL SECTION Materials. Methanol and glacial acetic acid were purchased from Mallinckrodt (Phillipsburg, NJ). Piperidine, imidazole, benzoquinoline, and Amberlite IRN77 hydrogen form were obtained from Sigma-Aldrich (St. Louis, MO). The siRNA [sense strand 5′OH-r(UGCGACAGGAGAUAGGCUG)d(TT)-3′OH, and antisense strand 5′OH-r(CAGCCUAUCUCCUGUCGCA)d(TT)-3′OH] targeting the ABCB1 gene sequence were purchased from QIAGEN (Valencia, CA). (32) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806–812. (33) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37 (7), 663–75. (34) Han, X.; Jian, M.; Breuker, K.; McLafferty, F. W. Science 2006, 314, 109– 112.

RNA samples were desalted either by repeated ethanol precipitations or by using a cation-exchange resin35 prior to mass spectrometry analysis. To perform ethanol precipitation, RNA samples were first dissolved in ultrapure water. Precipitation was carried out by first adding 10 M aqueous ammonium acetate to a RNA solution to bring the solution to 2.5 M in ammonium acetate, followed by the addition of 2.5 volumes of ice-cold ethanol. After incubating at -20 °C for at least 2 h, the RNA pellet was collected by centrifugation at room temperature for 40 min at 10 000 rpm. The supernatant was decanted, and the pellet was dried for 20 min in a centrifugal concentrator (SpeedVac, Savant UVS400, Holbrook, NY). The pellet was washed once with 200 µL of cold ethanol and once with 70% aqueous ethanol by vortexing. This was followed by centrifugation of the sample for 20 min at 10 000 rpm and then by drying the sample using SpeedVac. The above procedure was repeated two or three times. Following ethanol precipitation, the RNA pellet was stored at -80 °C. It was resuspended in water to a concentration of 100-200 µM as a stock solution just before mass spectrometric analysis. The desalting of RNA by the cation-exchange resin (Amberlite IRN77 hydrogen form) was carried out as described below. The cation-exchange resin was rinsed with DEPC-treated H2O and then mixed with siRNA stock solution of about 200 µM at room temperature for 10 min to reduce the cation adducts (Na+ or K+). For the generation of duplex siRNAs, the sense and antisense strand of siRNA were suspended in siRNA suspension buffer (100 mM potassium acetate, 30 mM HEPES-KOH, 2 mM magnesium acetate, pH 7.4) to 20 µM. The solution was heated at 90 °C for 1 min and then slowly cooled down to room temperature. The annealed siRNA sample was also desalted by cation-exchange resin treatment. RNA solutions for negative nanoelectrospray (nanoESI) were prepared by diluting the aqueous stock solutions to ca. 20 µM in 20/80 (v/v) 2-propanol/DEPC-treated water with the addition of 25 mM piperidine and 25 mM imidazole.36 Apparatus and Procedures. All experiments were performed using a prototype version of a QqTOF tandem mass spectrometer modified to allow for ion/ion reaction studies.37 A home-built pulsed dual nano-ESI source was coupled to the nanospray interface to produce ions of both polarities.38 The multiply deprotonated siRNA anions, [M - nH]n-, were generated directly via a nano-ESI emitter, whereas the singly protonated reagent cations were generated indirectly by ionizing the benzoquinoline vapor in between the curtain plate and orifice of the instrument via nano-ESI generated charged droplets. For proton-transfer ion/ ion reactions, the RNA anions were reacted with the reagent cations in Q2 collision cell. To perform ion trap CID, Q1-selected precursor ions were injected into Q2 at a relatively low kinetic energy, and a dipolar rf signal with frequency in resonance with the fundamental secular frequency of the precursor ions was then applied to one pair of the Q2 rods to induce ion trap CID. The fragment ions were then subjected to mass analysis by time-offlight (TOF). To facilitate interpretation of the product ion spectra from top-down analysis of intact siRNA, proton-transfer reactions (35) Huber, C. G.; Buchmeiser, M. R. Anal. Chem. 1998, 70, 5288–5295. (36) Greig, M.; Griffey, R. M. Rapid Commun. Mass Spectrom. 1995, 9, 97– 102. (37) Xia, Y.; Chrisman, P. A.; Erickson, D. E.; Liu, J.; Liang, X.; Londry, F. A.; Yang, M. J.; McLuckey, S. A. Anal. Chem. 2006, 78, 4146–54. (38) Xia, Y.; Liang, X.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 1750–1756.

Figure 1. Beam-type CID of siRNA duplex anions: (a) dissociation of the siRNA duplex [D siRNA]8- at a kinetic energy of 208 eV; (b) dissociation of the siRNA duplex [D siRNA]9- at a kinetic energy of 234 eV; (c) dissociation of the siRNA duplex [D siRNA]10- at a kinetic energy of 210 eV.

were employed to reduce the charge states of the fragment ions. A typical scan function for the proton-transfer ion/ion reaction of fragment ions consisted of steps such as RNA anion injection, isolation of ions of interest by Q1 in mass resolving mode, ion trap CID in Q2, cation injection to Q2, mutual cation/anion storage, and then mass analysis by TOF.39 A transmission mode proton-transfer ion/ion reaction could also be carried out by passing the reagent ions through the Q2 reaction chamber without mutual storage.40 RESULTS AND DISCUSSION Collisional Activation of siRNA Duplexes. Recently, the preservation of the duplex structure of siRNAs purified from the cells has been observed in an LC-MS setup. Information about the stability and pharmacokinetics of therapeutic siRNAs can be obtained from the masses of both the intact and the enzymatically processed siRNAs.41 The negative nano-ESI mass spectral results for the system studied here, which demonstrate the formation of gas-phase duplex anions, are provided as Supporting Information (see Figure S-1). The dissociation phenomena of DNA duplexes for different charge states and dissociation conditions have been reported.42,43 Tandem mass spectrometry of siRNA duplexes, however, has not been reported. Given the low-energy 5′ P-O backbone cleavage channel that operates for RNA anions but not for DNA anions, the dissociation behavior of RNA duplexes under collisional activation conditions is of interest. In this study, various charge states of siRNA duplexes were subjected to either beamtype or ion trap CID. As shown in Figure 1, the siRNA duplexes (39) Xia, Y.; Wu, J.; Londry, F. A.; Hager, J. W.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 71–81. (40) Liang, X.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2007, 18, 882–890. (41) Beverly, M.; Hartsough, K.; Machemer, L. Rapid Commun. Mass Spectrom. 2005, 19, 1675–1682. (42) Gabelica, V.; De Pauw, E. J. Am. Soc. Mass Spectrom. 2001, 13, 91–98. (43) Gabelica, V.; De Pauw, E. Int. J. Mass Spectrom. 2002, 219, 151–159.

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were predominantly dissociated into the individual sense and antisense siRNAs upon collisional activation in all charge states investigated. Hence, despite the possibility for 5′ P-O backbone cleavage, dissociation of the interstrand noncovalent bonds dominates, as is also noted for DNA duplexes. The partitioning of the total negative charges into the single-stranded counterparts is sequence-dependent, as is evident when the charges are asymmetrically distributed (e.g., from D8- to AS5- and S3- or AS3- and S5-). The sense strand consistently tends to compete more favorably for charge relative to the antisense strand. Therefore, the S5- ion is more abundant than the AS5- ion, and the S3- anion is less abundant than the AS3- anion in Figure 1a. A similar trend is observed in precursor ions of the two higher charge states (Figure 1, parts b and c). By increasing the translational energy in beam-type collisional activation or excitation amplitude in ion trap collisional activation, fragment ions from both strands were observed (data not shown). However, sequence information could not be readily derived because of the inability to distinguish the origin of the fragment ions from either strand. Ion Trap CID of Single-Stranded siRNAs. The effect of precursor ion charge state on the dissociation of multiply deprotonated gaseous nucleic acids has been reported in previous studies.44-47 To evaluate the effect of precursor ion charge on the dissociation of the siRNA anions, precursor ions of various charge states from each of the model siRNA strands were isolated in Q1 and subjected to ion trap CID in Q2. Similar to DNA anions, nucleobase loss is a facile dissociation channel from RNA anions, although the latter also show cleavage at the 5′ P-O backbone bonds. The competition between the neutral and charged nucleobase loss was observed to be sensitive to charge state. For example, the CID of the 5- precursor ion of the sense strand of siRNA (S siRNA) led primarily to neutral base losses and the c/yion series from 5′ P-O bond cleavages (see Supporting Information Figure S-2a). As precursor ion charge increased, charged base losses became increasingly competitive. Both neutral and charged base losses, for example, were observed from CID of the 10- of the S siRNA (Supporting Information Figure S-2b). Exclusive loss of the deprotonated adenine was found to be the dominant dissociation channel from CID of the 13- precursor ion of S siRNA (Supporting Information Figure S-2c). Similar phenomena were observed from CID of the antisense siRNA (AS siRNA). Generally, neutral base loss is favored at lower charge states; charged base loss is favored at higher charge states. A proton-bound dimer intermediate model, previously proposed to account for similar observations for the dissociation of DNA anions, is also consistent with the trends observed for RNA anions.44 The proton-bound dimer intermediate model consists of the nucleobase and a phosphodiester linkage. As the total charge of the ion increases, relief of electrostatic repulsion within the ion increasingly favors loss of the base as an anion. In addition, the effect of precursor ion charge state on the competition between the 5′ P-O bond cleavages and base losses has also been noted in multiply deprotonated RNA anions.47 The c/y-ion series from the 5′ P-O (44) McLuckey, S. A.; Vaidyanathan, G.; Habibi-Goudarzi, S. J. Mass Spectrom. 1995, 30, 1222–1229. (45) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2004, 237, 197–241. (46) Pan, S.; Verhoeven, K.; Lee, J. K. J. Am. Soc. Mass Spectrom. 2005, 16, 1853–1865. (47) Huang, T.; Kharlamova, A.; Liu, J.; McLuckey, S. A. J. Am. Soc. Mass Spectrom., in press.

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bond cleavages are more prevalent from precursor ions of lower charge states, generating the characteristic c/y-ion series for sequence characterization (see Supporting Information Figure S-2). As the precursor ion charge states increase, the base losses become dominant. Therefore, much less sequence information can be obtained from the higher charge states unless higher excitation amplitudes are applied, which generally lead to the subsequent 3′ C-O bond cleavages from the base loss ions. It is therefore desirable to dissociate the RNA anions of relatively low charge states in order to acquire sequence information. To develop a mass spectrometry based method for de novo RNA sequencing, it is desirable to generate complete and nonredundant sequence information from tandem mass spectrometry, while avoiding the formation of noninformative or possibly misleading fragmentation products. Most of the previous studies on gas-phase dissociation of RNA were conducted under beamtype collisional activation conditions. Complex spectra with fragment ions from all possible dissociation channels as well as products from multiple generation fragmentations were usually obtained. A complicated mixture of ions from several generations of cleavages complicates interpretation of the spectra. In part for this reason, tandem mass spectrometry has so far been limited to use in sequence verification of small enzymatically digested RNA or in mapping the post-transcriptional modification of RNAs with a known genome sequence.28-31 Figure 2 shows the beamtype CID results for the 5- antisense strand, [AS siRNA]5- at three laboratory collision energies (i.e., Figure 2, part a 140 eV, part b 190 eV, and part c 240 eV). At the lowest collision energy, the least extent of multiple fragmentations is noted but the efficiency of conversion of precursor ions to product ions is low. As the collision energy increases, a greater array of products are observed that include base losses, c-ions, y-ions, w-ions, (a-B)ions, as well as unidentified products that likely arise from sequential decomposition. In comparison to beam-type CID, ion trap CID is relatively more sensitive to the low-energy dissociation channels.48 The fact that the 5′ P-O bond cleavage is a relatively low-energy dissociation channel and is more or less independent of the identity of the nucleobases make complete sequencing of RNA molecules possible with largely one ion series (i.e., c/y ions). In addition, the increasing preference of base losses over 5′ P-O bond cleavages at higher charge states indicates that dissociation of precursor ions of relatively low charge states is more likely to generate predominant c/y-ion series with less competitive fragment ions from other channels.47 Ion trap collisional activation conditions were therefore explored with relatively low charge state precursor ions with the objective of establishing conditions that provide wide sequence coverage while minimizing spectral complexity. As shown in Figure 3a, ion trap CID of the AS siRNA anions (5-) at relatively low excitation amplitude predominantly led to dissociation through the lowest energy dissociation channels, neutral base losses and 5′ P-O bond cleavages. Full sequence coverage from nearly complete c/y-ion series, c1-c19 ions and y2-y20 ions, was obtained. Low-abundance w1-w3 ions from the 3′ C-O bond cleavage were also observed. Due to the relatively low fragment ion charge states and the relatively simple spectrum with the sequence informative ions mostly from single(48) McLuckey, S. A.; Goeringer, D. E. J. Mass Spectrom. 1997, 32, 461–474.

Figure 2. Beam-type CID product ion spectra of [AS siRNA]5- at laboratory collision energies of (a) 140, (b) 190, and (c) 240 eV.

Figure 3. Ion trap CID of the antisense siRNA anions [AS siRNA]5- under relatively low amplitude (a) 99.93 kHz, 480 mV, 100 ms and relatively high amplitude (b) 99.93 kHz, 800 mV, 100 ms conditions. The c/y-ion series labeled in panel a were not labeled in panel b to avoid figure congestion.

dissociation channels (5′ P-O bond cleavages), the interpretation of the spectrum is relatively straightforward. By gradually increasing the excitation amplitude, the dissociation rate of the precursor

ion could be increased, which increased the conversion of precursor ions to product ions. Up to a point, the product ion spectrum showed mainly an increase in the absolute abundances Analytical Chemistry, Vol. 80, No. 22, November 15, 2008

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of the c/y-ion series without increasing the spectral complexity due to the presence of other dissociation channels (data not shown). By further increasing the excitation amplitude, as shown in Figure 3b, the neutral base losses and the 5′ P-O bond cleavages remained the dominant dissociation channels but the a-B/w-ion series, which arise from the subsequent dissociation of the base loss ions [M - base]n- through 3′ C-O bond cleavages, became much more prominent. In the case of Figure 3b, in addition to the c/y-ion series, an extensive array of a-B/w ions was observed (w1-5,9,10, w14, a3-B, a4-B, a7-B, a11-B, and a12-B). Other backbone cleavages and secondary fragmentations were also observed at relatively low abundances. These dissociation channels contributed to the increase in baseline noise and peak congestion observed in Figure 3b and, therefore, make complete peak assignment difficult. Generally, in comparison to beam-type CID, much less secondary fragmentation was observed when similar fractions of precursor ions were dissociated (see Figure 2). For beam-type CID to achieve similar dissociation efficiency, extensive secondary fragmentation was usually observed due to the use of relatively high collision energies. Proton-Transfer Ion/Ion Reactions of RNA Anions with Benzoquinoline Cations. The appearance of multiply deprotonated or protonated ions is an inherent characteristic in ESI mass spectra of biopolymers, which can lead to spectral complexity, especially when a complex mixture is analyzed. The utility of ion/ ion reactions to simplify nucleic acid ESI mass spectra has been demonstrated.49,50 Several proton-transfer reagents for charge state manipulation of multiply charged DNA anions have been evaluated and reported in previous studies. Among them, protonated benzoquinoline cations have been demonstrated to simplify the ESI spectra of DNA mixtures via proton-transfer ion/ion reactions in a 3D ion trap. Minimal adduct formation or fragmentation of the precursor ions was observed. The ability to manipulate precursor ion charge states not only facilitates the study of complex mixtures but also allows for the tandem mass spectrometry of a wide range of precursor ion charge states. In this study, charge state manipulation of multiply deprotonated RNA anions was effected using a prototype version of a QqTOF tandem mass spectrometer modified to allow for ion/ion reaction studies. Multiply deprotonated single-stranded siRNA anions (5- to 10-) were generated by negative ion nano-ESI. To generate the reagent cations, benzoquinoline was placed inside of the curtain plate and the benzoquinoline vapor was then ionized by the positively charged droplets generated by nano-ESI. To effect proton-transfer ion/ion reactions, the multiply deprotonated siRNA anions and the protonated benzoquinoline reagent cations were reacted in Q2 in mutual storage mode38 or transmission mode.39 In comparison to the charge state distribution of siRNA anions from negative ion nano-ESI (see Supporting Information Figure S-3b), the charge state of each strand of siRNAs was reduced to mostly 1- and 2- in the post-ion/ion mass spectrum (Supporting Information Figure S-3c). Little adduct formation was observed in the reduced charge states. The molecular masses of both the sense and antisense strand of siRNA were readily determined. Noncovalent complexes, such as protein-protein complexes, drug-RNA complexes, and RNA-protein complexes, play im-

portant roles in the biological systems. The methods for the characterization of these complexes using mass spectrometry have been developed.51,52 Charge state manipulation of noncovalently bound siRNA duplexes was also demonstrated via proton-transfer ion/ion reactions. As shown in Supporting Information Figure S-4c, the isolated 7- of the siRNA duplexes [D siRNA]7- was chargereduced to [D siRNA]4- to [D siRNA]2- with no dissociation observed. One of the problems encountered in the dissociation of multiply charged precursor ions is the congestion of the product ions in a narrow m/z window, which limits the acquisition of sequence information when a high-resolution mass analyzer is not available. In the present study, each charge state of the sense and antisense strand of the siRNA anions was subjected to ion trap CID under various excitation amplitudes. Extended sequence coverage from the c/y-ion series was usually acquired. However, the peak overlap in some cases made charge state determination difficult, especially when precursor ions of higher charge states were subjected to dissociation. In addition, the spectral complexity increased when higher excitation amplitude was applied. To simplify the product ion spectrum from dissociation of multiply charged siRNA anions, ion/ion proton-transfer reactions were used to reduce the charge states of fragment ions to mostly 1- and 2-. Figure 4 shows the post-ion/ion ion trap CID spectrum of [AS siRNA]5- at a relatively low excitation amplitude. Complete sequence coverage from the c/y-ion series was obtained. Although the sequence information was comparable to the results from ion trap CID without ion/ion reaction (Figure 3a), difficulties with charge state ambiguity and peak congestion were resolved in the post-ion/ion ion trap CID spectrum. When higher excitation amplitudes were applied, peak assignment was much more difficult due to the increase in spectral complexity and baseline noise. The utility of ion/ion protontransfer reactions is especially evident in this scenario. Figure 5 shows the post-ion/ion ion trap CID spectrum of [AS siRNA]5at a relatively high excitation amplitude. Similar to the result from ion trap CID without ion/ion reaction (Figure 3b), complete sequence coverage from the c/y-ion series was obtained. However, significantly more sequence information from the lower abundant a-B/w-ion series was acquired. The sequence coverage of the antisense siRNAs from post-ion/ion ion trap CID is summarized in Figures 4d and 5d. Similar results were observed for the sense strand (data not shown). The results reported here suggest that the slow heating nature of ion trap CID is particularly useful for generating sequence information for anions of RNA oligomers at moderate to low charge states. The informative c/y-ion series can be more readily favored over the a-B/w-ion series using ion trap collisional activation than using beam-type collisional activation. The mechanism that gives rise to the former ions is generally less basedependent than the mechanism that leads to the latter ions. Beamtype collisional activation and ion trap collisional activation at relatively high amplitudes generate contributions from both ion series, which might be desirable in some cases. However, the information available from the two ion series is largely redundant

(49) McLuckey, S. A.; Wu, J.; Bundy, J. L.; Stephenson, J. L.; Hurst, G. B. Anal. Chem. 2002, 74, 976–984. (50) Wu, J.; McLuckey, S. A. Int. J. Mass Spectrom. 2003, 228, 577–597.

(51) Hanson, C. L.; Robinson, C. V. J. Biol. Chem. 2004, 279, 24907–24910. (52) Hofstadler, S. A.; Sannes-Lowery, K. A. Nat. Rev. Drug Discovery 2006, 5, 585–595.

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Figure 4. Post-ion/ion ion trap CID spectrum of [AS siRNA]5- with relatively low excitation amplitude applied (99.93 kHz, 480 mV). Panels a-c correspond to mass range m/z 300-1800, 1800-3300, and 3300-6800, respectively. (d) Summary of backbone cleavages resulting from post-ion/ion ion trap CID of the [AS siRNA]5- anions at relatively low excitation amplitude.

Figure 5. Post-ion/ion ion trap CID spectrum of [AS siRNA]5- with relatively high excitation amplitude applied (99.93 kHz, 800 mV). Panels a-c correspond to mass range m/z 300-1800, 1800-3300, and 3300-6800, respectively. (d) Summary of backbone cleavages resulting from post-ion/ion ion trap CID of the [AS siRNA]5- anions at relatively high excitation amplitude. Analytical Chemistry, Vol. 80, No. 22, November 15, 2008

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and contributions from further fragmentation, which also often contribute, can complicate spectral interpretation. CONCLUSIONS Most tandem mass spectrometry based methods for the characterization of RNAs have been limited to relatively small RNAs from enzymatic digestion (i.e., a bottom-up approach). The commonly used beam-type collisional activation method can efficiently dissociate RNAs; however, complex spectra with fragment ions from two dominant cleavage mechanisms as well as secondary dissociation can significantly hamper its utility in sequencing. In this study, tandem mass spectrometry applied to intact single strands of siRNA has been demonstrated to provide complete primary sequence information using a commercial quadrupole/TOF tandem mass spectrometer adapted for ion/ion reaction studies. Both single-stranded and duplex siRNAs were observed after annealing. Their dissociation behaviors from various charge states were investigated via collisional activation. The siRNA duplexes were predominantly dissociated through the breaking of noncovalent bonds and did not provide useful sequence information. The preferred dissociation channels of single-stranded siRNAs could be specifically directed to the 5′ P-O bond cleavages and neutral base losses when precursor ions of relatively low charge states were subjected to ion trap collisional activation at relatively low excitation amplitudes. As the excitation amplitude increased, the other sequence informative channels, such as 3′ C-O bond cleavages, began to contribute and could therefore be observed. Moreover, ion/ion proton-transfer reactions of RNA anions have been conducted using protonated benzoquinoline cations as the proton-transfer reagent. Charge reduction of an siRNA mixture and noncovalently bound siRNA duplexes has been demonstrated. The ability to manipulate RNA charge states in the gas phase allows the investigation of the gas-phase (53) Reid, G. E.; Shang, H.; Hogan, J. M.; Lee, G. U.; McLuckey, S. A. J. Am. Chem. Soc. 2002, 124, 7353–7362. (54) Liu, J.; Chrisman, P. A.; Erickson, D. E.; McLuckey, S. A. Anal. Chem. 2007, 79, 1073–1081.

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dissociation of RNAs of various charge states. In addition, the analysis of noncovalent complexes or RNAs from complex mixtures could be facilitated by ion/ion proton-transfer reactions through spectrum simplification, peak resolving, or gas-phase concentration via ion parking.53 Furthermore, to simplify the complex product ion spectrum, the multiply charged fragment ions can be charge-reduced to mostly singly and doubly charged species via proton-transfer ion/ion reactions with benzoquinoline cations. The resulting post-ion/ion product ion spectra allow the acquisition of complete sequence information from an originally unresolved CID spectrum. The degree of benefit to the use of ion/ion proton-transfer reactions for product ion charge state reduction is related to the performance characteristics of the mass analyzer.54 The connection between noncoding RNAs and important biological functions has drawn attention to the identification of human disease-related noncoding RNAs. However, most biochemical methods are incapable of identifying the post-transcriptional modifications, which frequently occur in noncoding RNAs. Mass spectrometry is a promising candidate for its sensitivity and efficiency in structural characterization of macromolecules. The combination of ion trap CID with ion/ion proton-transfer reactions, demonstrated in this study, provides a suitable platform for topdown analysis of these RNA molecules from complex mixtures. ACKNOWLEDGMENT This research was sponsored by the Purdue Research Foundation and the National Science Foundation under CHE-0808380. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 29, 2008. Accepted September 23, 2008. AC801331H