Anal. Chem. 1999, 71, 3436-3440
Multiplexed Screening of Neutral Mass-Tagged RNA Targets against Ligand Libraries with Electrospray Ionization FTICR MS: A Paradigm for High-Throughput Affinity Screening Steven A. Hofstadler, Kristin A. Sannes-Lowery, Stanley T. Crooke, David J. Ecker, Henri Sasmor, Sherilynn Manalili, and Richard H. Griffey*
Ibis Therapeutics, a Division of Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, California 92008
We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA-ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel. In recent years, electrospray ionization mass spectrometry (ESI-MS) has grown extensively as an analytical technique due to its broad applicability for analysis of macromolecules, including proteins, nucleic acids, and carbohydrates.1-4 One of the most significant developments in the field has been the observation, under appropriate solution conditions and analyte concentrations, of specific noncovalently associated macromolecular complexes that have been promoted into the gas phase intact.5-7 Recent examples include multimeric proteins,8 enzyme-ligand com(1) Bowers, M. T.; Marshall, A. G.; McLafferty, F. W. J. Phys. Chem. 1996, 100, 12897-12910. (2) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647R716R. (3) Biemann, K. Annu. Rev. Biochem. 1992, 61, 977-1010. (4) Crain, P. F.; McCloskey, J. A. Curr. Opin. Biotechnol. 1998, 9, 25-34. (5) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23. (6) Smith, R. D.; Bruce, J. E.; Wu, Q. Y.; Lei, Q. P. Chem. Soc. Rev. 1997, 26, 191-202.
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plexes,9 protein-DNA complexes,10 multimeric DNA complexes,11 and DNA-drug complexes.12 As evidenced by the abundance of recent publications, the implications and applications of these observations are far-reaching. The detection of noncovalent complexes has spurred the development of ESI-MS as a tool for drug discovery. Smith and co-workers have demonstrated that under competitive binding conditions in solution, ESI-MS measurements of enzyme-ligand mixtures yield gas-phase ion abundances that correlate with measured solution-phase dissociation constants (KD).13 They were able to rank the binding affinities of a 256-member library of modified benzenesulfonamide inhibitors to carbonic anhydrase. Several groups have demonstrated that levels of free and bound ligands and substrates can be quantified directly from their relative abundances as measured by ESI-MS and that these measurements can be used to quantitatively determine molecular-dissociation constants that agree with solution measurements. Jorgensen and co-workers have demonstrated that the relative ion abundance of noncovalent complexes formed between D- and L-tripeptides and vancomycin group antibiotics can be used to measure solution binding constants.14 Griffey and co-workers have shown that tandem ESI-MS methods can be used to determine the binding sites for small molecules that bind to RNA targets.15 These results suggest that ESI-MS has considerable potential for drug discovery. (7) Ens, W., Standing, K. G., Chernushevich, I. V., Ed. New Methods for the Study of Biomolecular Complexes. Proceedings of the NATO Advanced Research Workshop, June 16-20 1996, Alberta, Canada. In NATO ASI Series, Series C, 1998; 510; Kluwer: Dordrecht, The Netherlands, 1998. (8) Fitzgerald, M. C.; Chernushevich, I. V.; Standing, K. G.; Whitman, C. P.; Kent, S. B. H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6851-6856. (9) Ganguly, A. K.; Pramanik, B. N.; Huang, E. C.; Tsarbopoulos, A.; Girijavallabhan, V. M.; Liberles, S. Tetrahedron 1993, 49, 7985-7996. (10) Cheng, X.; Harms, A. C.; Goudreau, P. N.; Terwilliger, T. C.; Smith, R. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7022-7027. (11) Griffey, R. H.; Greig, M. J. Proc. SPIE-Int. Soc. Opt. Eng. 1997, 2985, 8286. (12) Gale, D. C.; Goodlett, D. R.; Light-Wahl, K. J.; Smith, R. D. J. Am. Chem. Soc 1994, 116, 6027-6028. (13) Cheng, X.; Chen, R.; Bruce, J. E.; Schwartz, B. L.; Anderson, G. A.; Hofstadler, S. A.; Gale, D. C.; Smith, R. D.; Gao, J.; Sigal, G. B.; Mammen, M.; Whitesides, G. H. J. Am. Chem. Soc 1995, 117, 8859-8860. (14) Jorgensen, T. J. D.; Roepstorff, P. Anal. Chem 1998, 70, 4427-4432. (15) Griffey, R. H.; Greig, M.; An, H.; Sasmor, H.; Manalili, S. J. Am. Chem. Soc. 1999, 121, 474-475. 10.1021/ac990262n CCC: $18.00
© 1999 American Chemical Society Published on Web 07/10/1999
Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) can resolve very small mass differences, providing determination of molecular mass with unparalleled precision and accuracy.16 Because each small molecule with a unique elemental composition carries an intrinsic mass label corresponding to its exact molecular mass, identifying closely related library members bound to a macromolecular target requires only a measurement of exact molecular mass. The target and potential ligands do not require radiolabeling, fluorescent tagging, or deconvolution via single-compound re-synthesis. Furthermore, adjustment of the concentration of ligand and target allows ESI-MS assays to be run in a parallel format under competitive or noncompetitive binding conditions. Signals can be detected from complexes with dissociation constants ranging from 100 nonbinding molecules does not adversely affect the signal of the expected 16S-paromomycin complex (data not shown), and identification of the “hits” is generally straightforward. Clearly, the use of neutral mass tags on a collection of RNA targets will not eliminate the possibility of overlapping complexes when large numbers of compounds are screened simultaneously; a key attribute of the neutral mass tags is that they allow the information to be dispersed over a wider m/z bandwidth. CONCLUSIONS In this work, we demonstrate that RNA targets with similar (or identical) molecular masses can be labeled with small neutral (22) Mei, H. Y.; Cui, M.; Heldsinger, A.; Lemrow, S. M.; Loo, J. A.; SannesLowery, K. A.; Sharmeen, L.; Czarnik, A. W. Biochemistry 1998, 37, 1420414212.
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molecules to measure binding between the targets and ligands using mass spectrometry. By screening multiple targets against ligand mixtures simultaneously, the information content of the assay is enhanced, resulting in a dramatic reduction in the number of analyses required. Although the increased complexity of the multisubstrate/ligand mixtures places high demands on the mass analyzer, this scheme facilitates the simultaneous analysis of numerous targets under identical solution conditions and ligand concentrations, further enhancing the high-throughput nature of the screening strategy and allowing direct comparisons of binding affinities for closely related targets. This concept of “rational” target design should also be applicable in studies of proteins that differ in amino acid sequence. ACKNOWLEDGMENT This work was supported in part by the Department of Commerce through a NIST Advanced Technology Program Grant (no. 97-01-0135) awarded to the Ibis Therapeutics Division of Isis Pharmaceuticals. Received for review March 5, 1999. Accepted May 17, 1999. AC990262N
