| This thesis focuses on proteomic methods development, analysis of proteomic response of Psychrobacter 273-4 and Escherichia coli in extreme environments and differential proteomic analysis of normal and O157:H7 Escherichia coli using two-dimensional HPLC combined with mass spectrometry. On-probe digestion was developed to perform protein digestion in 5 minutes with 5-18% improvement of protein sequence coverage. It was further integrated with monolithic column HPLC and off-line MALDI to identify proteins in breast cancer cells using only 120ng of proteins. Li-CHCA provides homogeneous spots on the MALDI plate when used with CHCA as a matrix. Lithium ions can not only be selectively attached to certain amino acid residues also selectively regulate the relative abundance of peaks in the MALDI spectrum, which allows more peptides detected in mass spectrometry analysis in certain fractions of E. coli. Four lines of Psychrobacter 273-4 cultured at 22°C and 4°C in the media with or without 5% sodium chloride were comparatively studied. Fifty-six proteins associated with given extreme environment, including five environment specific proteins, were identified. Combination effect of salinity and 4°C on protein expression has been observed. The mechanism of the adaptation to low temperature and salinity relies on the control of translation and transcription, the synthesis of proteins (chaperones) to facilitate RNA and protein folding, and the regulation of metabolic pathways. Six lines of E. coli propagated for 2,000 generations at 20°C were compared with their ancestors to provide the proteomic basis of evolutionary adaptation to low temperature. The differences of protein profiles between ancestor and descendants varied significantly with the origin and batch number of samples even they were developed from the same ancestor. This result suggests that the adaptation of E. coli to 20°C is achieved through multiple pathways. Analysis of differential protein expression between one normal and twoO157:H7 E. coli reveals that no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 could be distinguished from nonvirulent E. coli using a 2D UV map pattern as a "fingerprint". Several potential markers are identified for detection and treatment of O157:H7 infection. |
