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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Career Transition Award (K22)
Project #
5K22AI001748-02
Application #
6372689
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Taylor, Christopher E
Project Start
2000-09-01
Project End
2002-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
2
Fiscal Year
2001
Total Cost
$104,841
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Johnson, Jeffrey R; Meng, Fanyu; Forbes, Andrew J et al. (2002) Fourier-transform mass spectrometry for automated fragmentation and identification of 5-20 kDa proteins in mixtures. Electrophoresis 23:3217-23
Meng, Fanyu; Cargile, Benjamin J; Patrie, Steven M et al. (2002) Processing complex mixtures of intact proteins for direct analysis by mass spectrometry. Anal Chem 74:2923-9
Forbes, A J; Mazur, M T; Patel, H M et al. (2001) Toward efficient analysis of >70 kDa proteins with 100% sequence coverage. Proteomics 1:927-33
Meng, F; Cargile, B J; Miller, L M et al. (2001) Informatics and multiplexing of intact protein identification in bacteria and the archaea. Nat Biotechnol 19:952-7
Kelleher, N L (2000) From primary structure to function: biological insights from large-molecule mass spectra. Chem Biol 7:R37-45
Inamine, J M; Burdett, V (1985) Structural organization of a 67-kilobase streptococcal conjugative element mediating multiple antibiotic resistance. J Bacteriol 161:620-6