Improved T cell surfaceomics by depleting intracellularly labelled dead cells

Area of Science:

• Proteomics
• Cell Biology
• Biochemistry

Background:

• Plasma membrane (PM) proteins are crucial for cell function and therapeutic targeting.
• Current proteomic methods struggle to accurately capture PM proteins due to contamination.
• Cell surface biotinylation is a common enrichment technique but suffers from high intracellular protein contamination.

Purpose of the Study:

• To identify the source of intracellular protein contamination in cell surface biotinylation.
• To develop a method to reduce contamination and improve plasma membrane proteomics.
• To assess the impact of dead cell depletion on plasma membrane protein identification and accuracy.

Main Methods:

• Investigated intracellular labeling of non-viable cells using amine-reactive reagents.
• Implemented Annexin V staining and depletion of dead cells post-biotinylation.
• Analyzed plasma membrane protein enrichment, peptide counts, and iBAQ intensities before and after dead cell depletion.
• Examined changes in immature ER/Golgi glycoforms of CD11a and CD18.

Main Results:

• Non-viable cells, though a small percentage, accounted for a large proportion of labeled proteins.
• Dead cell depletion removed ~99% of intracellularly labeled cells.
• Plasma membrane protein intensity increased from 4% to 55.8% after dead cell depletion.
• Substantial depletion of intracellular, particularly nuclear, proteins was observed.
• Selective removal of immature ER/Golgi glycoforms of CD11a and CD18 occurred.

Conclusions:

• Intracellular labeling of non-viable cells is the primary cause of contamination in surface enrichment.
• Dead cell depletion is an effective strategy to reduce contamination and improve plasma membrane proteomics.
• This method enhances the sensitivity and accuracy of plasma membrane protein identification.