title: Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters.
abstract: This manuscript describes a new and general method to identify proteins localized into spatially restricted membrane microenvironments. Horseradish peroxidase (HRP) is brought into contact with a target protein by being covalently linked to a primary or secondary antibody, an antigen or substrate, a drug, or a toxin. A biotinylated tyramide-based reagent is then added. In the presence of HRP and hydrogen peroxide, the reagent is converted into a free radical that only diffuses a short distance before covalently labeling proteins within a few tens to hundreds of nanometers from the target. The biotinylated proteins can then be isolated by standard affinity chromatography and identified by liquid chromatography (LC) and mass spectrometry (MS). The assay can be made quantitative by using stable isotope labeling with amino acids in cell culture (SILAC) or isobaric tagging at the peptide level. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.|Cambridge Centre for Proteomics, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Johanna Susan
authors.initials: JS
authors.lastname: Rees
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Xue-Wen
authors.initials: XW
authors.lastname: Li
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Sarah
authors.initials: S
authors.lastname: Perrett
authors.affiliation: Cambridge Centre for Proteomics, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Kathryn Susan
authors.initials: KS
authors.lastname: Lilley
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Antony Philip
authors.initials: AP
authors.lastname: Jackson
mesh_terms: D000596:Amino Acids; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000031:analogs & derivatives*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D002853:Chromatography, Liquid; D006735:Horseradish Peroxidase; D006801:Humans; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D010455:Peptides; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014439:Tyramine / Q000031:analogs & derivatives*
chemical_list: D000596:Amino Acids; D000906:Antibodies; D008565:Membrane Proteins; D010455:Peptides; C111551:biotinyltyramide; D001710:Biotin; D006735:Horseradish Peroxidase; D014439:Tyramine
keywords: SPPLAT; protein microenvironments; proteomics; proximity; quantitative