Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome

Surface
biotinylation has been widely adapted in profiling the
cellular proteome associated with the plasma membrane. However, the
workflow is subject to interference from the cytoplasmic biotin-associated
proteins that compete for streptavidin-binding during purification.
Here we established a bioorthogonal conjugation-assisted purification
(BCAP) workflow that utilizes the Staudinger chemoselective ligation
to label and isolate surface-associated proteins while minimizing
the binding of endogenous biotin-associated proteins. Label-free quantitative
proteomics demonstrated that BCAP is efficient in isolating cell surface
proteins with excellent reproducibility. Subsequently, we applied
BCAP to compare the surface proteome of proliferating and senescent
mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified
and validated as a novel protein that is enhanced at the cell surface
of senescent MEFs. We expect that BCAP will have broad applications
in profiling cell surface proteomes in the future.