Proteome analysis of Escherichia coli K‐12 by two‐dimensional native‐state chromatography and MALDI‐MS

Abstract

SummaryTo identify proteins expressed in Escherichia coli K-12 MG1655 during exponential growth in defined medium, we separated soluble proteins of E. coli over two dimensions of native-state high-performance liquid chromatography, and examined the components of the protein mixtures in each of 380 fractions by peptide mass fingerprinting. To date, we have identified the products of 310 genes covering a wide range of cellular functions. Validation of protein assignments was made by comparing the assignments of proteins to specific first-dimension fractions to proteins visualized by two-dimensional gel electrophoresis. Co-fractionation of proteins suggests the possible identities of components of multiprotein complexes. This approach yields high-throughput gelindependent identification of proteins. It can also be used to assign identities to spots visualized by twodimensional gels, and should be useful to evaluate differences in expressed proteome content and protein complexes among strains or between different physiological states.

Quantitative N-Terminal Footprinting of Pathogenic Mycobacteria Reveals Differential Protein Acetylation

“…Our measurements may be biased due to the generally high abundance of proteins required for growth. 76 Indeed, proteins required for growth and protein synthesis are thought to have long half-lives in cells and NTA may promote protein stability. 6,7 However, we propose that our findings likely reflect mycobacterial physiology.…”

Differential enrichment of regulatory motifs in the composite network of protein-protein and gene regulatory interactions

“…It is observed earlier that not all genes are expressed in a given condition in an organism [26]. Genes coding for proteins that perform basic cellular functions are invariably expressed in all the conditions, and are therefore termed as essential genes.…”

Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives

“…To achieve a comprehensive understanding of cellular proteins, the limitations of 2D-PAGE should be overcome by adding other methods such as two-dimensional native and alternative types of chromatography that are independent of gels to separate individual protein components or peptides [28,29] and DNA micro array analysis. The new LC/MS/MS (using Q-TOF, Ion trap or TOF-TOF technology) technologies seem very promising.…”