Current Protocols in Protein Science · 2017 · 27 citations · 14 references
Biological Mass SpectrometryMolecular BiologyCell CultureAnalytical UltracentrifugationRedox BiologyOxidative StressProtein PurificationProteomic TechnologyHorseradish PeroxidaseBioanalysisQuantitative MethodImmunochemistryAnalytical BiotechnologyProteomicsBiochemistryBiotinylated ProteinsBiomolecular AnalysisMembrane BiologyNatural SciencesBiotechnologyProtein Mass SpectrometryMicrobiologyCellular BiochemistryMedicineProteomic Analysis
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.
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Andromeda: A Peptide Search Engine Integrated into the MaxQuant Environment
Jürgen Cox, Nadin Neuhauser, Annette Michalski et al. · Journal of Proteome Research · 2011 · 5.7K citations