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Updated: Jul 17, 2025

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

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Cell Surface Biotinylation Using Furan Cross-Linking Chemistry.

Esperanza Fernández^1,2, Laia Miret-Casals³, Annemieke Madder³

¹VIB Center for Medical Biotechnology, Ghent, Belgium.

Methods in Molecular Biology (Clifton, N.J.)

|September 4, 2023

Summary

This study introduces a novel surface biotinylation protocol using furan-biotin affinity purification. This method enhances the enrichment of plasma membrane proteins for more sensitive surface proteome analysis via mass spectrometry.

Keywords:

Affinity purification Furan-biotin LC-MS/MS Plasma membrane proteins Proteomics Surfaceome

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Area of Science:

Proteomics
Cell Biology
Biochemistry

Background:

Cellular surfaceome analysis is challenging due to low protein abundance and extraction difficulties.
Surface proteins are critical for cellular functions but are often lost during sample preparation.
Existing methods like surface biotinylation improve detection but can be further optimized.

Purpose of the Study:

To develop and validate a new, efficient protocol for enriching plasma membrane proteins.
To improve the sensitivity and accuracy of surface proteome mapping using mass spectrometry.
To overcome limitations of current surface protein extraction and enrichment techniques.

Main Methods:

Developed a novel surface biotinylation protocol utilizing furan-biotin affinity purification.
Applied the protocol to viable cells for selective labeling of surface membrane proteins.
Enriched biotinylated proteins using streptavidin beads, followed by trypsin digestion and LC-MS/MS analysis.

Main Results:

Successfully enriched plasma membrane proteins from cell surface.
Demonstrated improved sensitivity in mapping the surface proteome compared to conventional methods.
The furan-biotin protocol effectively addresses protein aggregation and low abundance issues.

Conclusions:

The furan-biotin affinity purification protocol offers a robust and sensitive approach for plasma membrane proteomics.
This method enhances the study of the cellular surfaceome, providing deeper insights into cell surface protein functions.
The protocol is a valuable tool for advancing mass spectrometry-based proteomic analyses of cell surface proteins.