Universal and robust bacterial phosphoproteomics analysis

In present work, we established a simple and robust protocol for universal bacterial phosphoproteomic analysis underlying the rationale of methanol-based LLE protein extraction and cleanup. Using the SDS lysis as the conventional protocol control, we carefully evaluated the efficiency of protein extraction and contaminants removal in terms of phosphopeptide enrichment, phosphopeptide identification and quantification in Listeria monocytogenes, a gram-positive bacterium. We found out the addition of urea, a chaotropic agent, facilitating the protein denaturation and then the protein-impurity dissociation, largely enhanced the protein extraction and sample purity. We further demonstrated the feasibility of this unified LLE platform for studying both gram-positive and gram-negative phosphoproteomes.