MASS-SPECTROMETRY AND EST-DATABASE SEARCHING ALLOWS CHARACTERIZATION OF THE MULTI-PROTEIN SPLICEOSOME COMPLEX

Many important cell mechanisms are carried our and regulated by multi- protein complexes, for example, transcription and RNA processing machi nery, receptor complexes end cytoskeletal structures. Most of these co mplexes remain only partially characterized due to the difficulty of c onventional protein analysis methods. The rapid expansion of DNA seque nce databases now provides whole or partial gene sequences of model or ganisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes(1). This approach has been demons trated in organisms whose genomes have been sequenced(2), such as budd ing yeast. Here we report the first characterization of an entire mamm alian multi-protein complex using these methods. The machinery that re moves introns from mRNA percursors-the spliceosome-is a large multi-pr otein complex(3,4). Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in prot ein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence t ag (EST) databases. Existing EST databases are thus already sufficient ly complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.