High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation...

Area of Science:

• Proteomics
• Single-cell analysis
• Microfluidics

Background:

• Mass spectrometry-based proteomics for single cells is limited by throughput and coverage.
• Existing methods struggle to analyze the proteome of individual mammalian cells effectively.
• Understanding single-cell protein expression is crucial for dissecting biological complexity.

Purpose of the Study:

• To develop a high-throughput platform for single-cell proteomics.
• To improve proteome coverage and analytical depth for individual cells.
• To enable unbiased characterization of single-cell proteome heterogeneity.

Main Methods:

• Integration of microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling.
• Multiplex analysis of single cell-sized protein quantities.
• Application to 72 single mammalian cells from epithelial, immune, and endothelial populations.

Main Results:

• Achieved proteome coverage of approximately 1,600 proteins per cell with high reproducibility (median CV 10.9%).
• Identified over 2,300 proteins across 72 single cells within 2 days of instrument time.
• Principal component analysis successfully segregated cells into three distinct populations based on proteomic profiles.

Conclusions:

• The developed platform significantly enhances throughput and proteome coverage for single-cell proteomics.
• It enables high-throughput, unbiased characterization of single-cell proteome heterogeneity.
• This technology advances the study of cellular diversity and function at the proteome level.