Champion, Matthew Maurice (2005-12). Functional proteomics in Escherichia coli. Doctoral Dissertation. | Thesis individual record

Cells respond to their environment with programmed changes in gene expression.
Cataloging these changes at the protein level is key towards understanding the physiology
of an organism. Multi-subunit and multi-protein complexes are also important and
pathogenic and physiologic processes. In order to identify expressed proteins and
potential protein complexes, we utilized a combination of non-denaturing
chromatography and peptide mass fingerprinting. This approach allows us to identify the
components of protein mixtures, as well as information lost in traditional proteomics,
such as subunit associations. Applying this methodology to cells at both mid-exponential
and stationary phase growth conditions, we identified several thousand proteins from
each cell-state of E. coli corresponding to hundreds of unique gene products. The copurification
of proteins when fractionated at varying pHs could suggest the components
of higher order complexes. This non-denaturing proteomic approach should provide
physiological data unavailable by other means. The components of several known
cellular complexes were also evident in this analysis. To characterize proteins associated
with nucleic acid binding, we also performed proteome analysis on log and stationary
phase cells grown in LB separated over heparin chromatography at neutral pH, which
enriches for these proteins. The complete analysis of these identifications is discussed.

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