Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling

Cell surface is the primary site
for sensing extracellular stimuli.
The knowledge of the transient changes on the surfaceome upon a perturbation
is very important as the initial changed proteins could be driving
molecules for some phenotype. In this study, we report a fast cell
surface labeling strategy based on peroxidase-mediated oxidative tyrosine
coupling strategy, enabling efficient and selective cell surface labeling
within seconds. With a labeling time of 1 min, 2684 proteins, including
1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular),
732 transmembrane proteins, and 81 cluster of differentiation antigens,
were identified from HeLa cells. By comparison with the negative control
experiment using quantitative proteomics, 500 (68%) out of the 731
significantly enriched proteins (p-value < 0.05,
≥2-fold) in positive experimental samples were cell surface-annotated
proteins. Finally, this technology was applied to track the dynamic
changes of the surfaceome upon insulin stimulation at two time points
(5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR,
CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated
(p-value < 0.01, ≥1.5-fold) after insulin
exposure by different mechanisms. We envision that this technique
could be a powerful tool to analyze the transient changes of the surfaceome
with a good time resolution and to delineate the temporal and spatial
regulation of cellular signaling.