PROTEOME SCANNING

University Of Illinois Urbana-Champaign, Champaign IL

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Abstract

The genome of strain M129 of the causative pathogen in greater than 10 percent of community-acquired pneumonia, Mycoplasma pneumoniae, has been completely sequenced and encodes 679 proteins. These genome-predicted proteins will be correlated with the entire primary structures of greater than 75 percent of those present in the M. pneumoniae proteome at a copy number greater than 10. By employing state-of-the-art mass spectrometry (MS) and an efficient "top down" strategy of protein analysis, events that generate a protein of different molecular composition than that predicted will be detected. These include sequence or reading frame errors, imprecise bioinformatics, co- or post- translational modification, and mutational or proteolytic strategies for antigenic variation. When covalent modification is indicated, the mass spectrometer can be used to assay for the modifying activity; this requires assembly of an automated MS- based assay system. This type of large scale protein analysis has not been demonstrated previously and relies on 2-D liquid separations instead of 2-D gels for protein separation and the unique capabilities of Electrospray Ionization coupled to Fourier-transform MS. Microbial genomes encode all possible virulence determinants, vaccine candidates, and potential drug and diagnostic targets, with most of these being proteins not DNA. Further, a completed genomic sequence establishes a basis for high throughput analysis of its gene products, a key part in understanding basic microbiology and identifying features for possible development of pathogen-specific detection methods and antimicrobial compounds. Thus, this study seeks to translate analytical advance into biological insight and establish a laboratory and career complementary to DNA sequencing and transcript profiling in the new era of biology.