Related Experiment Video
Updated: Jul 25, 2025
06:13
Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier
Published on: December 9, 2022
4.0K
Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for
Thy Truong1, Kei G I Webber1, S Madisyn Johnston1
1Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA.
Angewandte Chemie (International Ed. in English)
|June 28, 2023
Summary
Researchers developed a new method to quantify over 3,000 proteins from single cells using wide window acquisition (WWA). This technique enhances proteome coverage and enables rapid, label-free single-cell proteomic analysis.
Area of Science:
- Proteomics
- Cell Biology
- Analytical Chemistry
Background:
- Single-cell analysis presents significant challenges in proteomic depth and throughput.
- Label-free quantification requires highly sensitive and efficient methods for comprehensive protein identification.
Purpose of the Study:
- To develop and validate a novel proteomic strategy for quantifying thousands of proteins from single cells.
- To improve proteome coverage and efficiency in label-free mass spectrometry-based analyses.
- To compare proteome-wide expression changes in single cells with and without a specific autophagy gene knockout.
Main Methods:
- Integration of efficient sample preparation with ultra-low-flow liquid chromatography.
- Implementation of a new data acquisition and analysis scheme: wide window acquisition (WWA).
- WWA utilizes large isolation windows for co-isolation and co-fragmentation of precursor ions.
- Comparison of WWA performance against standard data-dependent acquisition (DDA).
Main Results:
- Quantification of >3,000 proteins per single-cell-sized aliquot using WWA.
- WWA increased MS2-identified proteins by ~40% compared to standard DDA.
- High proteome coverage (average 3,524 proteins) achieved with a 40-min LC gradient at ~15 nL/min.
- A 20-min gradient retained substantial proteome coverage (~90%).
- Identified 268 significantly differentially expressed proteins between atg9a knockout and WT single HeLa cells.
Conclusions:
- The developed WWA platform enables rapid, label-free quantification of >3,000 proteins from single cells.
- WWA significantly enhances proteome coverage and analytical efficiency in single-cell proteomics.
- Differential proteomic analysis revealed significant changes related to innate immunity, vesicle trafficking, and protein degradation upon atg9a knockout.
