| Popis: |
Bacteria need a quick and effective way to signal external natural or induced stimuli to internal responses. Part of this response is controlled by reversible protein phosphorylation, which is involved in e.g. gene transcription or secretion of virulence factors. Therefore, elucidation of the bacterial phosphoproteome is of utmost importance to aid understanding of bacterial virulence and antibiotic resistance. Unfortunately, the sub-stoichiometric nature of protein phosphorylation complicates the analysis and thus far hampers our understanding of bacterial phosphoproteomes. For the work described in my thesis I optimized sample preparation prior to Fe3+-IMAC enrichment and subsequent LC-MS/MS analysis enabling the in-depth analysis of the Staphylococcus aureus USA300 phosphoproteome. This allowed the identification of an unexpected high number of phosphorylation events, including the identification of new targets of the reportedly sole Serine-Threonine kinase and phosphatase in S .aureus. This high number of phosphorylation events indicates that the number of kinases in S .aureus is most likely still underestimated. Optimizing the sample preparation also allowed the identification of labile N-linked arginine phosphorylation, resulting in the presentation of the largest arginine phosphoproteome to date. Interestingly, arginine phosphorylation seems to be more prominent in S. aureus compared to other bacteria, indicating a potential important functional role. Validation of arginine phosphorylation by synthetic pArg peptides further showed that HCD is still the gold standard for phosphoproteomics. Lastly, we highlight that bacterial phosphoproteomics demands tailored protocols. Overcoming those remaining limitations should in future allow exhaustive and high-throughput analysis of bacterial phosphoproteomes, aiding a better understanding of bacterial signaling in virulence and resistance. |
