A Methanolic Urea-Enhanced Protein Extraction Enabling the Largest Bacterial Phosphorylation Resource.

Mass spectrometry (MS)-based phosphoproteomics analysis is a powerful approach for elucidating the regulatory roles of protein phosphorylation across all domains of life. However, bacterial phosphoproteomics still faces significant technical challenges due to the extremely low substoichiometry of phosphorylation evens and the structural complexity of bacterial cell envelopes, which impede efficient cell lysis, protein recovery, and purity. To address these obstacles, we developed Methanolic Urea-enhanced Protein Extraction (MUPE), a streamlined, detergent-free, solvent-based method that leverages the amphiphilic nature of methanol and the chaotropic properties of urea to enhance protein yield and lysis efficiency. Furthermore, MUPE seamlessly integrates with liquid-liquid extraction, enabling efficient protein purification without requiring sample transfer and complex manipulations. This workflow significantly improves phosphoproteome coverage and quantitative accuracy across Gram-positive and Gram-negative bacteria while minimizing sample input requirements. Our datasets substantially expand the known landscape of bacterial O-phosphorylation, revealing distinct phosphorylation preferences within bacterial signaling networks. Application of MUPE to Listeria monocytogenes under bile insult revealed extensive phosphorylation changes independent of protein expression, highlighting phosphorylation as a rapid and dynamic regulatory mechanism. Collectively, MUPE provides a robust and scalable platform for bacterial phosphoproteomic studies, advancing our understanding of phosphosignaling in the context of bacterial physiology and pathogenesis.