Liquid chromatography mass spectrometry-based proteomics of Escherichia coli single colony

Abstract

The Escherichia coli proteome is the most extensively characterized and studied of all prokaryotic proteomes. Despite this, large scale bacterial proteomics experiments performed on E. coli cells grown in liquid cultures have failed to identify key virulence factors thought to be important determinants in establishing bacterial infections. It seems likely that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth in biofilms, which can be crudely mimicked on solid media. This method describes a simple workflow to characterize the unique proteome signature of individual, isolated single colonies, using E. coli K12 strain grown on solid media as a model system. The workflow thus provides a means to explore the proteomes of minimally passaged clinical isolates of bacteria grown on primary culture plates and to identify both unique and differentially expressed proteins contained therein. Value of the method: - Simple mass spectrometry-based proteomics workflow to characterise the proteome of single colony forming units - Enables exploration of the proteomes of minimally passaged clinical isolates from primary culture plates - Identification of virulence factors expressed in true or mimicked biofilms that may be missed in liquid cultures Method name: E. coli single colony proteome analysis.

Keywords: Bacterial proteomics; E. coli; Mass spectrometry; Single colony; Virulence factors.

Graphical abstract

Fig. 1

Analysis of reproducibility between paired replicates. The multi scatter plot shows the log 2-transformed LFQ -values for protein groups identified in all six single colonies. The Pearson correlation coefficient is shown in each paired comparison and displayed in blue.

Fig. 2

Stacked bar graph showing the total MS and MS/MS recorded per Raw file of each single colony (SC). Orange bars showing the total MS recorded per SC, and their corresponding MS/MS indicated by stacked green bars. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Venn diagram of proteins across the six single colonies (SC1-6) demonstrating the overlap in number of protein between single colonies as well as the unique proteins identified in each single colony.