Multiplexed phosphoproteomics of low cell numbers using SPARCE.

Area of Science:

• Proteomics and cellular signaling analysis.
• Mass spectrometry applications in biological research.

Background:

• Global protein analysis is crucial for understanding cellular diversity and disease.
• Next-generation sequencing captures cellular heterogeneity but not downstream signaling.
• Phosphoproteomics is vital for signaling analysis but typically requires high cell input.

Purpose of the Study:

• To develop a method for sensitive phosphoproteomic analysis of rare cell populations.
• To overcome limitations in phosphoproteomics for low cell numbers.
• To enable unbiased protein-level analysis of signaling in limited samples.

Main Methods:

• Introduction of SPARCE (Streamlined Phosphoproteomic Analysis of Rare CElls) workflow.
• Integration of cell isolation, water-based lysis, and on-tip TMT labeling.
• Multiplexed phosphopeptide enrichment for enhanced quantification.

Main Results:

• SPARCE enhances labeling efficiency and phosphoproteome coverage compared to traditional methods.
• Successfully quantified phosphosite changes from as few as 1000 FACS-sorted glioblastoma stem cells.
• Demonstrated reliable analysis of rare cell populations.

Conclusions:

• SPARCE significantly advances phosphoproteomic analysis capabilities for rare cells.
• The workflow expands possibilities for signaling pathway investigation in limited biological samples.
• Enables deeper understanding of cellular diversity and disease mechanisms through protein modification analysis.