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

Johanna Susan Rees1, Xue‐Wen Li2, Sarah Perrett2, Kathryn Susan Lilley1, Antony Philip Jackson3

1 Cambridge Centre for Proteomics, University of Cambridge, Cambridge, 2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 3 Department of Biochemistry, University of Cambridge, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.27
DOI:  10.1002/cpps.27
Online Posting Date:  April, 2017
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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.

Keywords: SPPLAT; proteomics; proximity; protein microenvironments; quantitative

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: SPPLAT in Live Cells to Establish the Identity of Proteins in Cell Surface Protein Microenvironments
  • Support Protocol 1: Direct Infusion Mass Spectrometry to Assess Tyramide‐Biotin Coupling
  • Alternate Protocol 1: Quantitative SPPLAT in Cells by Metabolic Labeling of Cells in SILAC Media for the Quantitative Discrimination of Specific and Nonspecific Proteins
  • Support Protocol 2: Dialysis of Chicken Serum
  • Basic Protocol 2: Dynamic SPPLAT: To Establish the Identity of Proteins that Associate with a Target Over a Time Course, such as an Endocytic or Signaling Pathway
  • Support Protocol 3: Iron Loading of Transferrin
  • Basic Protocol 3: In Situ SPPLAT: Identification and Mapping of Proteins in Cryosectioned Tissue
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: SPPLAT in Live Cells to Establish the Identity of Proteins in Cell Surface Protein Microenvironments

  Materials
  • DT40 cells (ATCC® CRL‐2111)
  • RPMI media with 2 mM L‐glutamine (PAA) supplemented with 10% FBS (Life Technologies), 1% chicken serum (Sigma), 100 U/ml penicillin (Life Technologies), and 100 μg/ml streptomycin (Life Technologies).
  • PBS (see recipe)
  • Goat serum (Ultraclone Ltd.)
  • EZ‐link NHS‐SS‐biotin (Thermo Scientific)
  • Tyramine HCl (Sigma)
  • Sodium tetraborate decahydrate (Sigma)
  • Lysine
  • HRP‐conjugated goat anti‐chicken IgM (Bethyl Ltd.)
  • HRP‐conjugated goat anti‐rabbit IgG (BioRad)
  • Tyramide labeling buffer (see recipe)
  • Catalase (Sigma)
  • Antibody strip buffer (see recipe)
  • Cell lysis buffer (see recipe)
  • DNase I (Ambion)
  • Streptavidin (or neutravidin) agarose beads (Thermo Scientific)
  • Elution buffer (see recipe)
  • HRP‐conjugated streptavidin (Vector)
  • Colloidal Coomassie Blue (Life Technologies)
  • Liquid chromatography, mass spectrometry (LC/MS) grade water (Pierce)
  • 20 mM ammonium bicarbonate (NH 4HCO 3) in LC/MS‐grade water (Pierce)
  • 2 mM DTT (Sigma), freshly prepared
  • 10 mM iodoacetamide (Sigma), freshly prepared
  • 2 μg/ml sequencing‐grade bovine trypsin (Promega) in 50 mM ammonium bicarbonate (pH 8.0), freshly prepared
  • Acetonitrile (Sigma)
  • Formic acid (Sigma)
  • T25, T75, and T150 flasks with ventilation for CO 2
  • Low‐binding tubes (Eppendorf) to minimize loss of protein during purification steps
  • End‐over‐end rotator
  • 0.22‐μm filter units, syringe type (Millipore)
  • Scalpel
  • Glass vials for LC autosampler
  • Liquid chromatography system (e.g., nanoAcquity UPLC system, Waters Corp.)
  • Tandem mass spectrometer (e.g., LTQ Orbitrap Velos hybrid ion trap mass spectrometer, Thermo Scientific)
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, ), one‐dimensional SDS gel electrophoresis of proteins (unit 10.1; Gallagher, ), staining proteins in gels (unit 10.5; Echan and Speicher, ), immunoblot detection (unit 10.10; Gallagher, ), and in‐gel digestion of proteins (unit 11.3; Stone and Williams, )

Support Protocol 1: Direct Infusion Mass Spectrometry to Assess Tyramide‐Biotin Coupling

  Materials
  • Tyramide‐biotin preparation (Basic Protocol 1, steps 4 and 5)
  • 0.1% (v/v) formic acid
  • Syringe pump II (Harvard Apparatus)
  • SYNAPT G2 mass spectrometer (Waters Corp.)

Alternate Protocol 1: Quantitative SPPLAT in Cells by Metabolic Labeling of Cells in SILAC Media for the Quantitative Discrimination of Specific and Nonspecific Proteins

  Additional Materials ( )
  • 1% chicken serum, dialyzed ( protocol 4)
  • RPMI 1640 SILAC heavy medium containing 13C 6‐labeled L‐lysine and L‐arginine (K 6R 6) (Dundee Cell Products) and supplemented with 2 mM L‐glutamine (PAA Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma)
  • RPMI 1640 SILAC light medium containing non‐labeled L‐lysine and L‐arginine (K 0R 0) supplemented with 2 mM L‐glutamine (PAA Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma)

Support Protocol 2: Dialysis of Chicken Serum

  Materials
  • 10,000 Da cutoff dialysis cassettes (Thermo Scientific)
  • 2‐liter 0.15 M NaCl, ice cold
  • 100% chicken serum (Sigma)
  • Additional reagents and equipment for dialysis ( appendix 3B; Zumstein, 1995)

Basic Protocol 2: Dynamic SPPLAT: To Establish the Identity of Proteins that Associate with a Target Over a Time Course, such as an Endocytic or Signaling Pathway

  Materials
  • DT40 cells (ATCC® CRL‐2111)
  • RPMI media with 2 mM L‐glutamine (PAA) supplemented with 10% FBS (Life Technologies), 1% chicken serum (Sigma), 100 U/ml penicillin (Life Technologies), and 100 μg/ml streptomycin (Life Technologies)
  • RPMI 1640 SILAC heavy medium containing 13C 6‐labeled L‐lysine and L‐arginine (K 6R 6) (Dundee Cell Products) and supplemented with 2 mM L‐glutamine (PAA Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma)
  • RPMI 1640 SILAC light medium containing non‐labeled L‐lysine and L‐arginine (K 0R 0) supplemented with 2 mM L‐glutamine (PAA Laboratories), dialyzed 10% FBS (Dundee Cell Products), and dialyzed 1% chicken serum (Sigma)
  • Fe3+‐loaded, HRP‐conjugated apotransferrin (see protocol 6)
  • EZ‐link NHS‐LC‐biotin, non‐cleavable (Thermo Scientific)
  • Tyramine HCl (Sigma)
  • Sodium tetraborate decahydrate (Sigma)
  • 0.22 μm syringe‐filter units (Millipore)
  • 200 mM holo‐transferrin (human, iron‐loaded; Sigma)
  • Phosphate buffered saline (PBS) (see recipe)
  • Catalase (Sigma)
  • Tyramide‐labeling buffer (see recipe) containing 200 nM holo‐Transferrin
  • Cell lysis buffer (see recipe)
  • Streptavidin (or neutravidin) agarose beads (Thermo Scientific)
  • 0.1 M glycine, pH 2.5
  • 2 M NaOH
  • Mouse anti‐human transferrin receptor antibody (cross reactive with chicken; Life Technologies)
  • HRP‐conjugated goat anti‐mouse IgG (Biorad)
  • HRP‐conjugated streptavidin (Vector)
  • T25, T75 and T150 flasks with ventilation for CO 2
  • 37°C water bath
  • End‐over‐end rotator
  • Liquid chromatography system (e.g., nanoAcquity UPLC system, Waters Corp.)
  • Tandem mass spectrometer (e.g., LTQ Orbitrap Velos hybrid ion trap mass spectrometer, Thermo Scientific)
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, ), one‐dimensional SDS gel electrophoresis of proteins (unit 10.1; Gallagher, ), staining proteins in gels (unit 10.5; Echan and Speicher, ), immunoblot detection (unit 10.10; Gallagher, ), and in‐gel digestion of proteins (unit 11.3; Stone and Williams, )

Support Protocol 3: Iron Loading of Transferrin

  Materials
  • 1 mg HRP‐conjugated apotransferrin, human recombinant (Fitzgerald)
  • 10 mg/ml ferric ammonium citrate (Sigma) in PBS, pH 7.2
  • 1 liter PBS (see recipe), adjust to pH 7.2
  • 10,000 Da cutoff dialysis cassettes (Thermo)
  • Additional reagents and equipment for dialysis ( appendix 3B; Zumstein, 1995)

Basic Protocol 3: In Situ SPPLAT: Identification and Mapping of Proteins in Cryosectioned Tissue

  Materials
  • Cryosections of chosen material, 8 to 15 μm thick
  • PBS (see recipe or appendix 2E)
  • BSA (Sigma)
  • Saponin (Sigma)
  • Protease inhibitor cocktail tablets (Roche)
  • Antibody to target protein (HRP conjugated, if available, or use an HRP‐conjugated secondary)
  • Tyramide‐labeling reagents (see Basic Protocol 1); either cleavable or non‐cleavable linkers may be used: the permeability does not matter as sections are transverse
  • Fluorophore‐conjugated streptavidin (e.g., Alexa 647‐conjugated streptavidin, 1:1000)
  • Fluorophore‐conjugated secondary antibodies
  • Cell lysis buffer (see recipe)
  • Liquid chromatography system (e.g., nanoAcquity UPLC system, Waters Corp.)
  • Tandem mass spectrometer (e.g., LTQ Orbitrap Velos hybrid ion trap mass spectrometer, Thermo Scientific)
  • Cover slips
  • Confocal microscope
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, ), one‐dimensional SDS gel electrophoresis of proteins (unit 10.1; Gallagher, ), staining proteins in gels (unit 10.5; Echan and Speicher, ), immunoblot detection (unit 10.10; Gallagher, ), and in‐gel digestion of proteins (unit 11.3; Stone and Williams, )
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Figures

Videos

Literature Cited

Literature Cited
   Cox, J., Neuhauser, N., Michalski, A., Scheltema, R. A., Olsen, J. V., and Mann, M. 2011. Andromeda: A peptide search engine integrated into the maxquant environment. J. Proteome Res. 10:1794‐1805.
   Earnshaw, J.C. and Osbourn, J.K. 1999. Signal amplification in flow cytometry using biotin tyramine. Cytometry 35:176‐179.
   Echan, L. A. and Speicher, D. W. 2002. Protein detection in gels using fixation. Curr. Protoc. Protein Sci. 29:10.5:10.5.1‐10.5.18.
   Gallagher, S. 2001. Immunoblot detection. Curr. Protoc. Protein Sci. 4:10.10:10.10.1‐10.10.12.
   Gallagher, S. R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Protein Sci. 68:10.1:10.1.1‐10.1.44.
   Gross, A.J. and Sizer, I.W. 1959 The oxidation of tyramine, tyrosine, and related compounds by peroxidase. J. Biol. Chem. 234:1611‐1614.
   Hartman, N.C. and Groves, J.T. 2011. Signaling clusters in the cell membrane. Curr. Opin. Cell Biol. 23:370‐376.
   Jiang, S., Kotani, N., Ohnishi, T., Miyagawa‐Yamguchi, A., Tsuda, M., Yamashita, R., Ishiura, Y., and Honke, K. 2012. A proteomics approach to the cell‐surface interactome using the enzyme‐mediated activation of radical sources reaction. Proteomics 12:54‐62.
   Li, X.W., Rees, J.S., Xue, P., Zhang, H., Hamaia, S.W., Sanderson, B., Funk, P.E., Farndale, R.W., Lilley, K.S., Perrett, S., and Jackson, A.P. 2014. New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. J Biol. Chem. 289:14434‐14447.
   Olson, B. J. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Protein Sci. 48:3.4:3.4.1‐3.4.29.
   Ong, S.‐E. and Mann, M. 2006. Identifying and quantifying sites of protein methylation by heavy methyl SILAC. Curr. Protoc. Protein Sci. 46:14.9:14.9.1‐14.9.12.
   Rao, M. and Mayor, S. 2014. Active organization of membrane constituents in living cells. Curr. Opin. Cell Biol. 29:126‐132.
   Rees, J.S. and Lilley, K.S. 2011. Method for suppressing non‐specific protein interactions observed with affinity resins. Methods 54:407‐412.
   Rees, J.S., Lowe, N., Armean, I.M., Roote, J., Johnson, G., Drummond, E., Spriggs, H., Ryder, E., Russell, S., St Johnston, D., and Lilley, K.S. 2011. In vivo analysis of proteomes and interactomes using parallel affinity capture (iPAC) coupled to mass spectrometry. Mol. Cell Proteomics 10:M110.002386.
   Rhee, H.W., Zou, P., Udeshi, N.D., Martell, J.D., Mootha, V.K., Carr, S.A., and Ting, A.Y. 2013. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339:1328‐1331.
   Roux, K.J., Kim, D.I., Raida, M., and Burke, B. 2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196:801‐810.
   Stone, K. L. and Williams, K. R. 2004. Enzymatic digestion of proteins in gels for mass spectrometric identification and structural analysis. Curr. Protoc. Protein Sci. 38:11.3:11.3.1‐11.3.10.
Key References
  Li, X.W., Rees, J.S., Xue, P., Zhang, H., Hamaia, S.W., Sanderson, B., Funk, P.E., Farndale, R.W., Lilley, K.S., Perrett, S., and Jackson, A.P. 2014. New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. J. Biol. Chem. 289:14434‐14447.
  This key reference describes much of the historical development and optimization of SPPLAT for high throughput proteomics as well as the results and validation that can be achieved.
Internet Resources
  http://www.maxquant.org/
  MaxQuant freeware for the analysis of quantitative mass spectrometry data (Cox et al., ).
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