Profiling the Cellular Surfaceome by Furan-Based Protein Biotinylation.

The cellular surfaceome is crucial for cellular homeostasis and communication, with surface proteins acting as receptors, transporters, and enzymes. However, comprehensive analysis of the surfaceome is challenging due to the hydrophobic nature and low abundance of membrane proteins, leading to their underrepresentation in mass spectrometry-based proteomics data. This study exploits a novel method for profiling the cellular surfaceome using furan chemistry. We synthesized six furan-biotin compounds (FB1-FB6) and showed that FB1 reacts with lysine and cysteine. Upon furan activation by oxidation, FB1 exhibited the highest staining intensity and specificity for cell surface proteins. Pull-downs of biotinylated proteins further confirmed the efficiency of FB1 in enriching cell surface proteins, and FB1 was also found to reduce nonspecific labeling of intracellular proteins compared to biotinylation methods using N-hydroxysuccinimide esters. Our method thus provides a robust tool for unbiased, high-specificity proteomic studies of cell surfaceomes.