µPhos: a scalable and sensitive platform for high-dimensional phosphoproteomics.

Area of Science:

• Proteomics
• Cell Signaling
• Mass Spectrometry

Background:

• Phosphorylation analysis is key to understanding cell signaling.
• Current methods can be time-consuming and require large sample amounts.
• Defining cellular responses to perturbations requires sensitive phosphoproteomics.

Purpose of the Study:

• To develop an accessible and rapid phosphoproteomics platform (µPhos).
• To enhance sensitivity and quantitative reproducibility in phosphopeptide enrichment.
• To enable deep phosphoproteome analysis from limited sample quantities.

Main Methods:

• Developed µPhos (microPhos) for phosphopeptide enrichment from 96-well plates and small tissues.
• Minimized transfer steps and liquid volumes for increased efficiency.
• Utilized trapped ion mobility mass spectrometry for high-sensitivity quantification.

Main Results:

• µPhos achieves >90% selectivity and excellent quantitative reproducibility.
• Quantified ~17,000 Class I phosphosites from 20 µg and ~6200 from 1 µg of human cancer cell line.
• Revealed drug- and time-dependent response signatures in leukemia cells and spatial kinase activities in the mouse hippocampus.

Conclusions:

• µPhos significantly advances phosphoproteomics by enabling rapid, sensitive, and reproducible analysis.
• The platform supports sample-limited applications and large-scale perturbation studies.
• µPhos provides novel insights into cellular signaling and biological systems.