Mapping Nanoscale-To-Single-Cell Phosphoproteomic Landscape by Chip-DIA

Area of Science:

• Proteomics
• Cellular Signaling
• Mass Spectrometry

Background:

• Protein phosphorylation is vital for disease and drug development.
• Analyzing single-cell phosphoproteomics is challenging due to low signal.
• Understanding cellular signaling heterogeneity is key for precision medicine.

Purpose of the Study:

• To develop an ultrasensitive method for nanoscale-to-single-cell phosphoproteomic profiling.
• To map the phosphoproteomic landscape in lung cancer at single-cell resolution.
• To identify therapeutic targets and stratify patient subtypes.

Main Methods:

• Integration of a microfluidic phosphoproteomic chip (iPhosChip) with data-independent acquisition mass spectrometry (DIA-MS).
• A streamlined, all-in-one workflow for cell capture, imaging, and phosphoproteomic analysis.
• Library-based DIA-MS strategy for enhanced sensitivity and coverage.

Main Results:

• Chip-DIA achieved ultra-high sensitivity, detecting thousands of phosphopeptides from as few as 10 cells.
• The first single-cell phosphoproteomic landscape of lung cancer was revealed, including druggable sites.
• Heterogeneous signaling in EGFR-resistant lung cancer cells was identified, aiding in subtype stratification and therapy selection.

Conclusions:

• Chip-DIA enables unprecedented sensitivity and coverage for single-cell phosphoproteomics.
• This technology can unravel complex cellular signaling networks and guide precision oncology.
• The adaptable Chip-DIA platform can be applied to other post-translational modification omics studies.