Development of MS-based proteomics approaches to examine metabolic pathways and protein:protein interactions in microbial systems

The proteome is perhaps the most functional operating machinery for almost all biological processes, serving as the bridge to link the genome and phenotypes. The proteome undergoes dynamic changes in terms of the abundance or interactions, responding to the environmental stimuli. Understanding this dynamic of protein alterations is the key to delineate critical biological mechanisms. Mass-spectrometry-based proteomics is a powerful tool to systematically monitor the heterogeneous alterations of the proteome, including the changes of abundance, modifications and interactions. In this dissertation, a research project was built upon current proteomics approaches to solve the issues regarding to the sample preparation and data analysis that would help propel this approach to better address certain questions in environmental microbiology and molecular biology. In the first study, we have designed an experimental method that efficiently removes humic acids prior to proteolytic peptide measurement, thus addressing one major challenge associated with soil microbiome proteome extraction and subsequent MS measurement. The second study was aimed at employing advanced proteomics approaches to better understand the microbial drivers of environmental mercury processes by using two model organisms: Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132. Our results elucidated the global proteome impacts caused by the deletion of mercury methylation essential genes hgcAB and revealed that deletion of hgcAB genes did not show significant impact on the microbial response to mercury addition. The third study focused on optimizing MS-based proteome approaches for characterizing protein-protein interactions. By coupling the rapid crosslinking procedure, affinity enrichments and high-performance MS measurements, protein-protein interaction can be captured and interrogated in a living cell by a time-resolved manner. Overall, the methods and results presented in this dissertation not only provides an enhanced sample preparation methodology for intractable samples (such as soils), but also demonstrates how this systems-biology approach can be utilized to characterize the basics of microbial physiology at a global proteome level as well as drilling down into specific proteins interactions. The optimized methods and experimental/bioinformatics techniques described in this dissertation should be broadly extendable to proteome characterization/protein interaction examination in various systems