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High-Throughput Label-free Single-Cell Proteomics Enabled by Multicolumn NanoLC with a 5-min Cycle Time
Ryan Kelly1, Chao Wang1, Hsien-Jung Lin1
1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.
Research Square
|December 3, 2025
Summary
A new multicolumn nanoLC-MS platform enables high-throughput single-cell proteomics (SCP) by reducing run times to 5 minutes. This breakthrough allows for the analysis of thousands of cells per study, offering deep proteome coverage and biological insights.
Area of Science:
- Proteomics
- Mass Spectrometry
- Cellular Biology
Background:
- Mass spectrometry (MS)-based single-cell proteomics (SCP) provides high-resolution proteome analysis.
- Current nanoLC-MS workflows have low throughput due to long separation gradients.
- This limits the application of SCP in large-scale studies.
Purpose of the Study:
- To develop a high-throughput multicolumn nanoLC-MS platform for SCP.
- To enable the analysis of thousands of single cells within a single study.
- To maintain deep proteome coverage and biological interpretability.
Main Methods:
- Implementation of a multicolumn nanoLC-MS system with 5-minute separation windows.
- Operation at ~100 nL/min flow rate with 100% duty cycles.
- Analysis of over 4,000 samples, including RAW264.7 macrophages.
Main Results:
- Achieved throughput of ~288 single cells per day (SPD).
- Identified ~3,200 proteins per single HeLa cell, comparable to longer-gradient methods.
- Demonstrated accurate quantification across ~6,000 proteins and distinguished cellular proteomes.
- Revealed LPS-induced markers and macrophage activation pathways.
Conclusions:
- The developed platform is robust, scalable, and suitable for high-throughput SCP.
- Enables thousands of single-cell analyses in a single study.
- Maintains deep proteome coverage and biological interpretability for cellular heterogeneity studies.
