Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins

Comprehensive profiling of the cell-surface
proteome has been challenging
due to the lack of tools for an effective and reproducible way to
isolate plasma membrane proteins from mammalian cells. Here we employ
a proximity-dependent biotinylation approach to label and isolate
plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BioID. The lipid-modified
BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of
the plasma membrane, where it effectively biotinylated plasma membrane
proteins. Biotinylated proteins were then affinity-purified and analyzed
by mass spectrometry. Our analysis demonstrates that combining conventional
sucrose density gradient centrifugation and Plasma Membrane-BioID
is ideal to overcome the inherent limitations of the identification
of integral membrane proteins, and it yields highly pure plasma components
for downstream proteomic analysis.