Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane‐Bound Protein Clusters

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.

[1]  D. Speicher,et al.  Protein Detection in Gels Using Fixation , 2018, Current protocols in protein science.

[2]  M. Rao,et al.  Active organization of membrane constituents in living cells. , 2014, Current opinion in cell biology.

[3]  K. Lilley,et al.  New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay , 2014, The Journal of Biological Chemistry.

[4]  S. Carr,et al.  Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging , 2013, Science.

[5]  Brian Burke,et al.  A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells , 2012, The Journal of cell biology.

[6]  K. Honke,et al.  A proteomics approach to the cell‐surface interactome using the enzyme‐mediated activation of radical sources reaction , 2012, Proteomics.

[7]  J. Groves,et al.  Signaling clusters in the cell membrane. , 2011, Current opinion in cell biology.

[8]  K. Lilley,et al.  Method for suppressing non-specific protein interactions observed with affinity resins. , 2011, Methods.

[9]  Irina M. Armean,et al.  In Vivo Analysis of Proteomes and Interactomes Using Parallel Affinity Capture (iPAC) Coupled to Mass Spectrometry , 2011, Molecular & Cellular Proteomics.

[10]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[11]  J. Markwell,et al.  Assays for Determination of Protein Concentration , 2007, Current protocols in protein science.

[12]  M. Mann,et al.  Identifying and Quantifying Sites of Protein Methylation by Heavy Methyl SILAC , 2006, Current protocols in protein science.

[13]  S. Gallagher One‐Dimensional SDS Gel Electrophoresis of Proteins , 1995, Current protocols in molecular biology.

[14]  Enzymatic Digestion of Proteins in Gels for Mass Spectrometric Identification and Structural Analysis , 2004, Current protocols in protein science.

[15]  Immunoblot detection. , 2001, Current protocols in protein science.

[16]  J. Osbourn,et al.  Signal amplification in flow cytometry using biotin tyramine. , 1999, Cytometry.

[17]  I. Sizer,et al.  The oxidation of tyramine, tyrosine, and related compounds by peroxidase. , 1959, The Journal of biological chemistry.