E. coli proteomics

The use of CIEF in combination with FTICR has been demonstrated in an analysis of the E. coli proteome (Jensen et al., 1999). For these experiments, E. coli was grown in a medium depleted of rare isotopes in order to increase the mass measurement accuracy. The high abundance isotopes are present at approximately 98.89% 12C, 99.63% 14N and 99.985% H. For peptides, the presence of rare isotopes does not significantly change the spectra but with undigested proteins, mass accuracy can be limited by the broadened distribution of ions of any given protein due to the incorporation... 

Vollmer, M., Nagele, E., Horth, P. (2003). Differential proteome analysis two-dimensional nano-LC/MS of E. coli proteome grown on different carbon sources. J. Biomol. Tech. 14, 128-135. 

Thomas GH. Completing the E. coli proteome a database of gene products characterised since the completion of the genome sequence. Bioinformatics 1999 15 860-861. 

Taniguchi, Y. et al (2010) Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells Science, 329 (5991), 533-538. 

E. coli proteome by mass spectrometry. Anal. Chem. 2001, 73, 4063 70. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Fluorescein-5-maleimide also has been used to study the assembly dynamics of mycobacterium tuberculosis (Chen et al., 2007), to study monomers and dimers of nhaa Na+/H+ antiporter of E. coli (Rimon et al., 2007), and to investigate the regulation of the protein disulfide proteome by mitochondria (Yang et al., 2007). 

Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines (1) Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation,... 

The probes labeled purified LacZ, a glycosidase expressed in E. coli, as weh as several structurally unrelated glycosidases in complex proteomes [114, 115]. 

In P. aeruginosa, despite considerably lower levels of LasR when compared to the E. coli system, the same protocol resulted in detection of a band by in-gel fluorescence, which could be excised and the identification of LasR achieved by proteomic MS methods with 32% total sequence coverage. The methodology was also extended to allow analysis by flow cytometry. In this way, the authors were able to demonstrate that cell density had an effect on LasR labelling. As expected, fluorescence increased with cell density up to the point at which quorum was achieved (around OD600 = 6), beyond which fluorescence began to weaken. 

Ecogene University of Miami School of Medicine E. coli K-12 genome and proteome sequences, including extensive gene bibliographies (http //www. ecogene. org/3.0/)... 

Two-dimensional gel databases are located on the Internet. One for S. ceruisiae is found at www.proteome.com and one for both S. ceruisiae and E. coli is found at http /yexpasy. hcuge.ch/ch2d/. 

Phytochemistry 62,837-849,2003 Nobeli, I., Ponstingl, H., Krissinel, E.B., and Thornton, J.M., A strncture-based anatomy of the E.coli metabolome, J. Mol. Biol. 334, 697-719, 2003 Parsons, L. and Orban, J., Structural genomics and the metabolome combining computational and NMR methods to identify target hgands, Curr. Opin. Drug Discov. Devel. 7, 62-68, 2004 Soloviev, M. and Finch, P, Peptidomics bridging the gap between proteome and metabolome, Proteomics 6, 744-747, 2006. 

Savage DF, Anderson CL, Robles-Cohnenares Y, Newby ZE, Stroud RM. Cell-free complements in vivo expression of the E. coli membrane proteome. Protein Sci. 2007 16 966-976. Wiener MC. A pedestrian guide to membrane protein crystallization. Methods 2004 34 364-372. 

Table 2 Percentage of charged amino acids and (G - - C) content of 10 hyperthermophilic archaea (A), 2 hyperthermophilic bacteria (B), and mesophilic bacteria E. coli. A strong prevalence of lysine over arginine in proteomes of hyperthermophiles is obtained for nine organisms. A bold font marks the exception from the general trend...

The power of the currently available methods for proteome mining can be illustrated with data reported on the identification of 800 proteins from 50 milhon E. coli K12 cells using multiple fractionation, RPLC-MS-MS, and various ways of database searching [37]. With MASCOT searching, 754 proteins were identified from 1326 peptides and 2167 MS-MS spectra, which means on average 1.75 peptides and 2.87 spectra per protein. Abont 17% of the E. coli K12 proteome was covered in this way. 

In another study, related to the E. coli K12 proteome, by another group [37], the tryptic digest was first fractionated in 40 fractions by RPLC. Next, Met-containing peptides in these fractions were oxidized by HjOj. These modified proteins were separated from the bulk unmodified proteins by RPLC and fractionated in 8 fractions. These fractions were subsequently analysed by nano-LC-MS-MS on a Q-TOF instrument operated under DDA. After the first RPLC run, an exclusion hst was generated from peptides identified in a MASCOT search of the data, and the sample was then remn. Following this procedure, 754 proteins were identified, which is about 34% of the expressed E. coli K12 proteome. 

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