Mass Spectrometry-Based Proteomic Analysis of Selected Bacteria from the Human Gut Microbiome
Human gut bacteria live in a dynamic environment, constantly adapting their proteomes to changes like pH and nutrient availability. This study investigates how selected members of the human gut microbiome (HGM) respond to such conditions using mass spectrometry-based proteomics. Initially, the study evaluated two quantification methods—bottom-up label-free quantification (LFQ) and tandem mass tag (TMT)—in Bacteroides thetaiotaomicron. Both methods showed comparable results, indicating that this bacterium primarily alters the abundance of proteins involved in the machinery required to utilize the provided carbon sources The LFQ approach was then applied to examine proteomic changes in B. thetaiotaomicron, Blautia producta, and Bifidobacterium longum in response to different environmental pH levels. Distinct and concurrent alterations in pathway-related and stress-associated proteins were identified, including histidine biosynthesis in B. producta, nitrogen metabolism in B. thetaiotaomicron, and inositol carbohydrate metabolism in B. thetaiotaomicron and B. producta. The third project focused on identifying novel proteins, specifically short open reading frame-encoded peptides (SEP), in B. producta. The combined bottom-up and top-down proteomics analyses identified a total of 45 SEP, including previously reported SEP (BP1 to BP14). Their production varied based on environmental factors like media, pH, and supplements. The last project optimized a protocol for isolating extracellular vesicles (OMVs) from E. coli. A proteoform-directed top-down analysis, including a discovery-based open modification search, identified several potential post-translational modifications.
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