Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type...

Area of Science:

• Proteomics
• Cellular Biology
• Mass Spectrometry

Background:

• Single-cell proteomics (SCP) is crucial for understanding cellular heterogeneity but faces challenges in reproducible quantification and proteome depth.
• Existing methods struggle to identify thousands of proteins per cell with sufficient depth for complex biological analysis.

Approach:

• Developed a fully automated SCP workflow using the cellenONE picolitre dispensing robot and the proteoCHIP EVO 96.
• Integrated the proteoCHIP EVO 96 with the Evosep One chromatographic system for in-line desalting and separation.
• Utilized the Bruker timsTOF Ultra mass spectrometry instrument for high-sensitivity protein identification.

Key Points:

• The automated workflow reproducibly identifies up to 4,000 proteins per single HEK-293T cell.
• Evosep One separation improves protein identifications twofold compared to standard HPLC.
• Identified over 50 ubiquitin ligases and key signaling proteins in LPS-perturbed THP-1 cells.

Conclusions:

• This advanced SCP workflow provides unprecedented proteome depth for single cells.
• The proteoCHIP EVO 96 and timsTOF Ultra combination enables the study of complex biological phenomena beyond cell-type classification.
• The method offers a robust and reproducible solution for high-throughput single-cell proteomic analysis.