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

J Proteome Res. 2016 Feb 5;15(2):647-58. doi: 10.1021/acs.jproteome.5b01066. Epub 2016 Jan 12.


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.1-8 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.9-11 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.

Keywords: PIGS; aminooxy-biotin; biotin; cell surface; comparative analysis; plasma membrane; shotgun proteomics; silica beads; tunicamycin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Biotinylation
  • Cell Line, Tumor
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Chromatography, Liquid / methods
  • Humans
  • Mass Spectrometry / methods
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Proteome / genetics
  • Proteome / metabolism*
  • Proteomics / methods*
  • Reproducibility of Results
  • Tunicamycin / pharmacology


  • Anti-Bacterial Agents
  • Membrane Proteins
  • Proteome
  • Tunicamycin