Sulfo-NHS-LC-LC-Biotin (Biotin-XX-SSE), a biotin reagent, is employed for labeling proteins on the external leaflet of intact exosomes and features an extended spacer arm between the biotin and the amine-reactive linker. This elongated linker is designed to mitigate steric hindrance, thereby enhancing labeling efficiency on the densely populated exosome surface [1] [2].

Sulfo-NHS-LC-LC-Biotin was used for biotinylation of exosome proteins by mixing 200 μg of intact exosomes with 10 mM Sulfo-NHS-LC-LC-Biotin at room temperature for 30 minutes. The conditions considered were: (a) using an excess of Sulfo-NHS-LC-LC-Biotin for complete saturation of exposed lysine residues and potential N-termini, (b) the sulfonate group preventing reagent penetration of the exosomal membrane, (c) a 30.5 angstrom spacer arm enhancing biotinylation of proteins in their natural conformation, and (d) a 452 Da increase in mass for amino acids labeled with Sulfo-NHS-LC-LC-Biotin. Post-incubation, excess Sulfo-NHS-LC-LC-Biotin was removed using a 10 KDa MWCO filtration device [2]. Rat aortic endothelial cells (RAEC) were surface modified in suspension with 1 mM Sulfo-NHS-LC-LC-biotin for 10 minutes, then pelleted and resuspended in PBS [3].