Figure 3 Expression of lacZ and male. mRNA (dashed) and protein (solid) dynamics for periplasmic maltose-binding protein/malE (red) and β-galactosidase/lacZ (blue) upregulated during glucose-lactose diauxie (time 0).
Using the clustering function for large click here datasets, clara, from the R cluster package , the dataset could be broadly divided into groups of up- and downregulated proteins, along with proteins that do not change measurably as a function of the diauxic shift. The FTICR-ion trap cluster provided comprehensive label-free quantitative proteomic data with sufficient throughput for an arbitrary number of conditions or time points and biological replicates (here about 30), allowing a global study of protein expression dynamics in E. coli. With this instrument platform, proteomics data such as that presented here can be routinely generated in less than 48 h. To illustrate
changes in metabolic pathways, the protein expression data was mapped onto KEGG metabolic pathways and changes in level of expression indicated by color (Figure 4). Most proteins in the same pathways EPZ015938 as β-galactosidase were also markedly upregulated, leading to a global activation of the galactose pathway responsible for channeling lactose into the glycolytic pathway. Other metabolic pathways changed to a lesser degree, as measured by protein (enzyme) abundance. Figure 4 Protein expression mapped onto KEGG pathway. The protein expression profiles mapped onto the galactosidase metabolic pathways
highlights changes in metabolism when shifting from glucose to lactose as primary carbon source. The measured changes in enzyme (protein) abundance were converted to color and mapped onto KEGG pathways. Upregulated proteins are marked in green, downregulated in red, and unchanged in yellow. Conclusions We have reproduced the textbook glucose-lactose diauxie experiment in E. coli using a state-of-the-art method for quantitative proteomics using a novel mass spectrometry platform, the FTICR-ion ZD1839 trap cluster. In each of three experiments the onset of diauxie occurred at approximately the same cell density and the duration of diauxic shift was also similar. The identified and individually quantified peptides were collected into quantitative protein measurements, which were visualized and compared using tools developed in-house. Through kind assistance from KEGG it is now possible to upload color codes for a whole list of quantified proteins on any metabolic pathway overview (the R learn more program for generating the color codes from protein abundance ratios is available from the authors). We could confirm that the most strongly induced enzymes belong to the pathway responsible for glucose and lactose metabolism. The FTICR-ion trap cluster in combination with the appropriate visualization tools makes an efficient approach for investigation of protein expression dynamics.