Development of Mass Spectrometry methods for direct analysis of proteins in Microbes

Du, Yuying (2025). Development of Mass Spectrometry methods for direct analysis of proteins in Microbes. University of Birmingham. Ph.D.

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Abstract

Mass spectrometry (MS) is a powerful technique used to analyse proteins. A specific version, native liquid extraction surface analysis (LESA) mass spectrometry (MS) allows us to study proteins in their natural form, providing information on their tertiary and quaternary structure directly from different types of samples. In this thesis, this technology was developed for the analysis of proteins from living colonies of microorganisms, including bacteria and yeast. This design enables a deeper understanding of microbial proteomes in their native state, which is essential for studying biological processes, discovering new drug targets, and improving biotechnological applications.
To improve the approach of detecting different native proteins, methods were designed combining native LESA MS with various technologies, including electroporation (which helps extract proteins from cells), a filtering method named high-field asymmetric waveform ion mobility spectrometry (FAIMS), colony washing and a specialised particle system known as styrene maleic acid lipid particles (SMALPs).
This work demonstrates that the combination of native LESA MS and electroporation is effective for detecting native intact proteins in Escherichia coli K12 and Saccharomyces cerevisiae living colonies. Proteins were identified by use of a variety of fragmentation techniques. In some cases, use of FAIMS filtering improved detection, allowing for a more detailed analysis of proteins in yeast Saccharomyces cerevisiae.
The integration of native LESA MS and prior colony washing enabled detection of outer membrane proteins in gram-negative bacteria. Native LESA MS was also applied to the analysis of two membrane proteins (ZipA and Sav1855) encased in SMALPs. The results show detection of truncated versions of the proteins.
This research demonstrates the potential of LESA MS for studying proteins directly from living microorganisms, which could be valuable for relative disease research and drug development.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):