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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
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A proximity proteomics pipeline with improved reproducibility and throughput.
Xiaofang Zhong1,2,3, Qiongyu Li1,2,3, Benjamin J Polacco1,2,3
1Quantitative Biosciences Institute (QBI), University of California, San Francisco, San Francisco, CA 94158, USA.
Biorxiv : the Preprint Server for Biology
|April 24, 2023
Summary
We developed a scalable proximity labeling pipeline using automated biotinylation and mass spectrometry for efficient spatial proteome analysis. This method enhances throughput and reproducibility for studying cellular protein interactions.
Area of Science:
- Cellular and Molecular Biology
- Proteomics
- Biochemistry
Background:
- Proximity labeling (PL) coupled with mass spectrometry (MS) is crucial for mapping spatial proteomes in living cells.
- Existing methods require extensive hands-on time and can lack quantitative reproducibility for large-scale studies.
Approach:
- Developed a scalable PL pipeline with automated 96-well plate enrichment of biotinylated proteins.
- Integrated an optimized data-independent acquisition (DIA) MS method to enhance sample throughput and quantitative accuracy.
- Applied the pipeline to map subcellular proteomes and investigate dynamic protein interaction networks.
Key Points:
- Significantly increased sample throughput and improved reproducibility of protein identification and quantification.
- Successfully delineated subcellular proteomes of endosomes, lysosomes, Golgi apparatus, and plasma membrane.
- Investigated temporal changes in the 5HT2A serotonin receptor interactome upon serotonin activation.
- Demonstrated pipeline adaptability for CRISPR-based gene knockout studies with reduced sample input.
Conclusions:
- The presented scalable PL pipeline enhances throughput and reproducibility for proximity proteomics.
- This universally applicable approach facilitates the study of protein interactions across diverse cellular compartments and experimental conditions.
