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Updated: Jul 30, 2025
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Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT
Published on: May 1, 2017
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HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics
Yiran Liang1, Thy Truong1, Aubrianna J Saxton1
1Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States.
Analytical Chemistry
|May 11, 2023
Summary
This study combines stable isotope labeling of amino acids in cell culture (SILAC) and tandem mass tag (TMT) labeling to significantly increase throughput for single-cell proteomics (SCP) analysis. This novel approach enables higher-throughput single-cell proteome profiling.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biotechnology
Background:
- Single-cell proteomics (SCP) offers deep biological insights but is limited by low throughput.
- Existing methods like isobaric and isotopic labeling enhance throughput individually.
Purpose of the Study:
- To develop a method combining isobaric and isotopic labeling for multiplicative scaling of SCP throughput.
- To characterize the efficiency and proteome coverage of this combined labeling strategy.
Main Methods:
- Combined two-plex SILAC and TMT labeling for multiplexed SCP.
- Utilized a custom nested nanowell chip for nanoliter sample processing.
- Performed liquid chromatography-mass spectrometry (LC-MS) analysis with optimized cycle times.
Main Results:
- Achieved analysis of up to 28 single cells per LC-MS run.
- Demonstrated a throughput of ~280 single cells per day, scalable to ~700 cells per day.
- Characterized labeling efficiency and proteome coverage under various conditions.
Conclusions:
- Combined SILAC and TMT labeling significantly enhances SCP throughput.
- The developed method provides a scalable solution for high-throughput single-cell proteome analysis.
- This advancement facilitates deeper biological investigations using SCP.
