Abstract
Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes1. This approach has been demonstrated in organisms whose genomes have been sequenced2, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors — the spliceosome — is a large multi-protein complex3,4. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.
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Acknowledgements
We thank U. Ryder and F. Caufrier for help with the spliceosome column. The other members of the Protein & Peptide group, especially A. Shevchenko, M. Wilm and A. V. Podtelejnikov, are acknowledged for fruitful discussions and technical help. Work in M.M.'s laboratory at EMBL was supported by generous grants from the German Technology Ministry and from Glaxo Wellcome. A.I.L. is a Wellcome Trust Principal Research Fellow.
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Neubauer, G., King, A., Rappsilber, J. et al. Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet 20, 46–50 (1998). https://doi.org/10.1038/1700
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DOI: https://doi.org/10.1038/1700
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