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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

This summary is machine-generated.

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.

Keywords:

biotinylation glycosylation intracellular pool plasma membrane surfaceome

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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.