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

Area of Science:

• Proteomics
• Biotechnology
• Cell Biology

Background:

• Single-cell proteomics aims to quantify proteins in individual cells.
• Technological advances improve sensitivity and throughput but face quantification challenges.
• Reproducible protein quantification at the single-cell level is crucial for biological insights.

Purpose of the Study:

• To develop and validate a novel sample preparation chip for single-cell proteomics.
• To enhance protein identification and quantification in single cells.
• To enable deeper proteome profiling for complex biological investigations.

Main Methods:

• Development of the proteoCHIP EVO 96 sample preparation chip.
• Integration with the Evosep One liquid chromatography system.
• Analysis using Bruker timsTOF and timsTOF Ultra mass spectrometry platforms.
• Application to HEK-293T cells for proteome profiling.

Main Results:

• The proteoCHIP EVO 96 combined with timsTOF Ultra achieved up to 4000 protein group identifications per single cell.
• An average of 3500 protein groups were identified per single HEK-293T cell.
• The workflow demonstrated a 4-order-of-magnitude dynamic range.
• Over 50 E3 ubiquitin-protein ligases and key regulatory proteins were identified.

Conclusions:

• The proteoCHIP EVO 96 and timsTOF Ultra platform significantly advances single-cell proteomics capabilities.
• This workflow provides sufficient proteome depth for studying complex biological processes beyond cell-type classification.
• The method enables reproducible quantification and identification of thousands of proteins in single cells.