A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations

Plasma membrane (PM) proteins contribute
to the identity of a cell,
mediate contact and communication, and account for more than two-thirds
of known drug targets.− In the past years, several protocols for the proteomic profiling
of PM proteins have been described. Nevertheless, comparative analyses
have mainly focused on different variations of one approach.− We compared sulfo-NHS-SS-biotinylation, aminooxy-biotinylation,
and surface coating with silica beads to isolate PM proteins for subsequent
analysis by one-dimensional gel-free liquid chromatography mass spectrometry.
Absolute and relative numbers of PM proteins and reproducibility parameters
on a qualitative and quantitative level were assessed. Sulfo-NHS-SS-biotinylation
outperformed aminooxy-biotinylation and surface coating using silica
beads for most of the monitored criteria. We further simplified this
procedure by a competitive biotin elution strategy achieving an average
PM annotated protein fraction of 54% (347 proteins). Computational
analysis using additional databases and prediction tools revealed
that in total over 90% of the purified proteins were associated with
the PM, mostly as interactors. The modified sulfo-NHS-SS-biotinylation
protocol was validated by tracking changes in the plasma membrane
proteome composition induced by genetic alteration and drug treatment.
Glycosyl­phosphatidylinositol (GPI)-anchored proteins were depleted
in PM purifications from cells deficient in the GPI transamidase component PIGS, and treatment of cells with tunicamycin significantly
reduced the abundance of N-glycoproteins in surface
purifications.