Development of liquid extraction surface analysis mass spectrometry for protein analysis in microbial colonies

[摘要] Liquid extraction surface analysis (LESA) is an ambient mass spectrometry (MS) technique applied for the analysis of proteins from a range of biological substrates. The work described in this thesis focuses on the development of LESA MS-based approaches for the analysis of proteins from microbial biofilms directly on solid nutrient media. Both microbiological research applications as well as clinical applications for the identification of pathogens were considered during development. The reproducibility of LESA sampling on model surfaces and bacterial colonies was investigated. Complementary imaging methods were used to characterise the effects of LESA sampling on the surface of colonies. Sampling protocols were optimised to access proteins from Gram-positive bacteria. Identification of over 40 proteins by LESA MS was subsequently demonstrated for Gram-positive and Gram-negative clinical isolates; de novo sequencing of a novel protein from an unknown strain was also achieved. Characteristic protein masses were used to differentiate closely related species of streptococci. The capabilities of LESA MS for protein identification and characterisation were expanded by the coupling of high-field asymmetric waveform ion mobility spectrometry (FAIMS) into the workflow, overcoming several limitations encountered in the earlier stages of the project. The final challenge involved the sampling of yeast colonies, not ordinarily amenable to lysis by solvents compatible with LESA due to the presence of a thick cell wall. A device was constructed to allow the lysis of yeast colonies directly on agar plates by application of high voltage electric pulses to the colonies prior to LESA sampling. The extraction and identification of proteins in three yeast species was subsequently demonstrated.

[发布机构] University:University of Birmingham;Department:School of Biosciences

[关键词] Q Science;QR Microbiology