title: A Surface Biotinylation Strategy for Reproducible Plasma Membrane Protein Purification and Tracking of Genetic and Drug-Induced Alterations.
abstract: 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.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: Katrin
authors.initials: K
authors.lastname: Hörmann
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: Alexey
authors.initials: A
authors.lastname: Stukalov
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: Leonhard X
authors.initials: LX
authors.lastname: Heinz
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.|Center for Physiology and Pharmacology, Medical University of Vienna , 1090 Vienna, Austria.
authors.forename: Giulio
authors.initials: G
authors.lastname: Superti-Furga
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: Jacques
authors.initials: J
authors.lastname: Colinge
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , 1090 Vienna, Austria.
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019921:Biotinylation; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D008565:Membrane Proteins; D020543:Proteome; D014415:Tunicamycin
keywords: PIGS; aminooxy-biotin; biotin; cell surface; comparative analysis; plasma membrane; shotgun proteomics; silica beads; tunicamycin