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Universal and robust bacterial phosphoproteomics analysis

ABSTRACT: 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.

ORGANISM(S): Escherichia Coli Cellular Organisms

SUBMITTER: Miao-Hsia Lin

PROVIDER: PXD055159 | JPOST Repository | Mon Aug 04 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	20240202_LM_Tris50_phos_1.raw	Raw
	20240202_LM_Tris50_phos_2.raw	Raw
	LM_1MUrea_phos_1.raw	Raw
	LM_1MUrea_phos_2.raw	Raw
	LM_Tris50_phos_2.raw	Raw

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Publications

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

Wu Pei-Shan PS Chen Ting-An TA Chen Bo-Yu BY Ishihama Yasushi Y Lin Miao-Hsia MH

Molecular & cellular proteomics : MCP 20250624 8

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 ...[more]

PMID: 40571162

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