RESUMEN
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.
RESUMEN
Mass spectrometry-based single-cell proteomic analysis has recently gained momentum and is now an emerging area with established protocols and promising results. Traditional proteomic studies, especially involving bacteria, have been limited to suspension cultures with large protein yields. Such studies, however, remain population centered with the uniqueness of individual responses to environmental challenges becoming diluted. To enable bacterial single-colony proteomics, we describe a quantitative mass spectrometry-based protocol to isolate and analyze the proteome of a single mycobacterial colony from 7H10 media, with growth supplements for optimal growth. Following protein purification and digestion, tryptic peptides are analyzed by UHPLC coupled to a hybrid Q Exactive mass spectrometer. Raw data were analyzed using the MaxQuant Suite, and downstream statistical analysis was performed using Perseus software. A total of 7805 unique peptides and 1387 proteins were identified. Data are available via ProteomeXchange with identifier PXD018168. In this chapter, we identify steps most prone to sample loss and describe measures of alleviation that allows the preservation of protein yield and boosts quantitative power while increasing reproducibility, of "very limiting samples."