Single-Cell Proteomics Reveals Proteome Remodeling and Cellular Heterogeneity During NGF-Induced PC12 Neuronal...

Area of Science:

• Proteomics
• Cellular Biology
• Neuroscience

Background:

• Single-cell proteomics is crucial for understanding cellular heterogeneity in dynamic processes.
• Analyzing fragile, adherent neuronal models presents significant technical challenges for single-cell proteomics.

Purpose of the Study:

• To develop and apply an optimized single-cell proteomics workflow for characterizing proteome remodeling during nerve growth factor (NGF)-induced differentiation of PC12 cells.
• To investigate the heterogeneity and asynchronous nature of proteomic changes during neuronal differentiation.

Main Methods:

• Implemented gentle cell dissociation, anti-aggregation strategies, and thermal inkjet cell dispensing for accurate single-cell isolation.
• Utilized n-dodecyl-β-d-maltoside (DDM) to enhance recovery of membrane-associated and low-solubility proteins.
• Coupled the workflow with LC-ion mobility-mass spectrometry for high-throughput proteomic quantification (2,000-3,000 proteins per cell).

Main Results:

• Revealed progressive and heterogeneous proteome remodeling during NGF-induced PC12 cell differentiation.
• Identified distinct subpopulations with unique proteomic states and asynchronous differentiation trajectories using dimensionality reduction and clustering.
• Demonstrated that key proteins for differentiated neuronal states are underrepresented in bulk analyses but enriched in specific single-cell subpopulations.

Conclusions:

• The optimized workflow enables robust single-cell proteomics in neuronal systems.
• NGF-induced PC12 differentiation involves heterogeneous and divergent proteomic states, highlighting the limitations of bulk proteomics.
• Single-cell proteomics provides a deeper understanding of asynchronous cellular differentiation processes.