A Rapid One-Pot Workflow for Sensitive Microscale Phosphoproteomics.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Phosphoproteomics sensitivity has historically lagged behind single-cell proteomics due to sample complexity and input requirements.
• Existing methods often involve lengthy sample preparation and substantial sample amounts, limiting microscale applications.

Purpose of the Study:

• To develop a rapid, sensitive, and robust one-pot phosphoproteomics workflow (SOP-Phos) for microscale analysis.
• To integrate SOP-Phos with data-independent acquisition mass spectrometry (DIA-MS) to improve phosphoproteomic identification and quantification.

Main Methods:

• A novel one-pot protocol (SOP-Phos) using sodium deoxycholate lysis, direct trypsinization, and TiO2 bead enrichment in a single tube.
• Integration with n-dodecyl β-d-maltoside precoated tubes to minimize adsorptive losses.
• Application of data-independent acquisition mass spectrometry (DIA-MS) for enhanced data acquisition.

Main Results:

• SOP-Phos workflow completed in 3-4 hours, achieving 1.4-fold higher phosphopeptide identification coverage.
• SOP-Phos-DIA demonstrated >90% specificity, enhanced sensitivity, low missing values (<1%), and improved reproducibility (8-10% CV).
• Identified thousands of phosphopeptides from low microgram cell lysates and thousands of cells, enabling mapping of key signaling sites like EGFR autophosphorylation.

Conclusions:

• SOP-Phos-DIA is an efficient and robust protocol for microscale phosphoproteomic analysis.
• The workflow significantly improves sensitivity and reduces sample input requirements.
• Demonstrated feasibility in lung cancer models, providing mechanistic insights into EGFR-TKI resistance.