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Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells
Christofer Daniel Sánchez1, Aswath Balakrishnan1, Blake Krisko1
1Department of Pathology and Laboratory Medicine, University of California, Irvine, California, USA.
Molecular & Cellular Proteomics : MCP
|December 26, 2025
Summary
High intracellular protein contamination in plasma membrane proteomics is caused by dead cells. Depleting dead cells post-biotinylation significantly improves plasma membrane protein identification and accuracy.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- The plasma membrane (PM) is crucial but challenging to study using proteomics.
- Effective cell surface enrichment is vital for accurate quantitative surfaceomics.
- Current biotinylation methods yield high intracellular protein contamination.
Purpose of the Study:
- To identify the source of intracellular protein contamination in PM proteomics.
- To develop a method to reduce this contamination.
- To improve the accuracy and sensitivity of plasma membrane proteomics.
Main Methods:
- Investigated cell surface biotinylation using amine-reactive reagents.
- Identified labeling of non-viable cells as a major contamination source.
- Implemented Annexin V-based dead cell depletion post-labeling.
Main Results:
- Dead cells (5±2%) accounted for 90% of labeled proteins in T cells.
- Dead cell depletion removed ~99% of intracellularly labeled cells.
- Plasma membrane protein intensity increased from 4% to 55.8% after depletion.
- Depletion reduced intracellular protein contaminants, especially nuclear proteins.
- Immature ER/Golgi glycoforms of CD11a and CD18 were selectively removed.
Conclusions:
- Non-viable cells are the primary source of intracellular contaminants in PM proteomics.
- Dead cell depletion is an effective strategy to enhance plasma membrane proteomics.
- This method improves sensitivity, accuracy, and data quality for PM protein analysis.
