# Improved T Cell Surfaceomics by Depleting Intracellularly Labelled Dead Cells

## TL;DR

Removing dead cells after labeling improves the accuracy of cell surface protein studies by reducing contamination from intracellular proteins.

## Contribution

Dead cell depletion after biotinylation significantly enhances surfaceomics by eliminating intracellular contaminants from nonviable cells.

## Key findings

- Dead cell depletion increases plasma membrane protein intensity from 4% to 55.8%.
- Dead cells account for 90% of labeled proteins in T cell preparations despite being only 5% of the total.
- Intracellular pools of PM proteins are removed without affecting surface-resident proteins.

## Abstract

Although the plasma membrane (PM) is among the most biologically important and therapeutically targeted cellular compartments, it is among the most challenging to faithfully capture using proteomic approaches. The quality of quantitative surfaceomics data depends heavily on the effectiveness of the cell surface enrichment used during sample preparation. Enrichment improves sensitivity for low abundance PM proteins and ensures that the changes detected reflect PM expression changes rather than whole cell changes. Cell surface biotinylation with PM-impermeable, amine-reactive reagents is a facile, accessible, and unbiased approach to enrich PM proteins. However, it results in unexpectedly high contamination with intracellular proteins, reducing its utility. We report that biotinylating human cells with amine-reactive reagents intracellularly labels a small but reproducible population of nonviable cells. Although these dead cells represent only 5 ± 2% of the total, we find that in T cell preparations the dead cells account for 90% of labelled proteins. Depleting Annexin V positive dead T cells postlabelling removes ∼99% of the intracellularly labelled cells, resulting in markedly improved PM identifications, peptide counts, and intensity-based absolute quantification intensities. Correspondingly, we found substantial depletion of intracellular proteins, particularly of nuclear origin. Overall, the cumulative intensity of PM proteins increased from 4% to 55.8% with dead cell depletion. Finally, we demonstrate that immature ER/Golgi glycoforms of CD11a and CD18 are selectively removed by dead-cell depletion. We conclude that high intracellular labelling of nonviable cells is the major source of intracellular protein contaminants in amine-reactive surface enrichment methods and can be reduced by dead-cell depletion postlabelling, improving both the sensitivity and accuracy of PM proteomics.

•Amine-labelling based surfaceomics suffers from high intracellular contamination.•Non-viable cells are the major source of intracellular contaminants in Jurkat cells.•Dead cell depletion after biotinylation removes ∼99% of dead cells.•Cell surface protein intensity increased >10-fold with depletion.•Intracellular pools of PM proteins are removed but not surface-resident proteins.

Amine-labelling based surfaceomics suffers from high intracellular contamination.

Non-viable cells are the major source of intracellular contaminants in Jurkat cells.

Dead cell depletion after biotinylation removes ∼99% of dead cells.

Cell surface protein intensity increased >10-fold with depletion.

Intracellular pools of PM proteins are removed but not surface-resident proteins.

Surfaceomics reliant on amine-reactive biotinylation suffers from high contamination due to non-viable cells that are intensely intracellularly labeled. Even at low frequencies, these cells disproportionately contaminate surfaceomics studies with highly abundant intracellular proteins. Depletion of Annexin V-positive dead cells after labelling removes ∼99% of these biotinhigh cells, markedly improving plasma membrane (PM) protein enrichment and sensitivity. This method increased cumulative PM protein intensity from 4% to 55.8% and selectively removed intracellular pools of PM proteins while retaining surface-resident pools.