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Oral Sessions
Day 2, June 23（Mon.） 15:55-16:10
Room C (Top of Yaima)
- 2C-O3-1555
A Methanolic Urea-enhanced Protein Extraction Enabling the Largest Bacterial Protein O-Phosphorylation Atlas
(1NTU, 2Kyoto Univ., 3NIBIOHN)
Pei-Shan Wu1,2, Ting-An Chen1, Yasushi Ishihama2,3, oMiao-Hsia Lin1,2
Understanding protein O-phosphorylation dynamics is crucial for elucidating regulatory mechanisms across all life forms. However, bacterial phosphoproteomics faces challenges due to the lower substoichiometry of phosphorylated proteins compared to eukaryotes and the rigid and complex envelope structures. To facilitate system-wide exploration, we developed a faster and robust organic solvent method instead of detergent, termed as Methanolic Urea-enhanced Protein Extraction (MUPE). It markedly improved phosphoproteome depth, coverage, and reproducibility in both Gram-positive and Gram-negative bacteria, resulting in the most comprehensive bacterial phosphoproteome atlas (~21000 O-phosphosites:11,375 phosphosites in </I>L. monocytogenes</I>, 963 in </I>S. aureus</I>, 4,055 in </I>B. subtilis</I>, 3,412 in </I>E. coli</I> and 1,410 in </I>P. aeruginosa</I>) and allowing us to identify preferences for basophilic and acidophilic residues in bacterial phosphorylation. Notably, this simpler and fluent MUPE procedure demonstrated its applicability to determine the rapid phosphosignaling mechanism for stress sensing and adaptation, distinct from protein expression. These findings suggest that MUPE is a powerful tool that will greatly advance our understanding of bacterial phosphosignaling, particularly in the context of bacterial physiology and pathogenesis.