Metabolic Labeling with Noncanonical Amino Acids and Visualization by Chemoselective Fluorescent Tagging

Susanne tom Dieck1, Anke Müller2, Anne Nehring1, Flora I. Hinz1, Ina Bartnik1, Erin M. Schuman1, Daniela C. Dieterich2

1 Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Frankfurt, Germany, 2 Otto‐von‐Guericke‐University Magdeburg, Medical Faculty, Institute for Pharmacology and Toxicology, Magdeburg, Germany
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.11
DOI:  10.1002/0471143030.cb0711s56
Online Posting Date:  September, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Fluorescent labeling of proteins by genetically encoded fluorescent protein tags has enabled an enhanced understanding of cell biological processes but is restricted to the analysis of a limited number of identified proteins. This approach does not permit, e.g., the unbiased visualization of a full proteome in situ. We describe here a fluorescence‐based method to follow proteome‐wide patterns of newly synthesized proteins in cultured cells, tissue slices, and a whole organism. This technique is compatible with immunohistochemistry and in situ hybridization. Key to this method is the introduction of a small bio‐orthogonal reactive group by metabolic labeling. This is accomplished by replacing the amino acid methionine by the azide‐bearing methionine surrogate azidohomoalanine (AHA) in a step very similar to classical radioisotope labeling. Subsequently, an alkyne‐bearing fluorophore is covalently attached to the group by “click chemistry”—a copper(I)‐catalyzed [3+2]azide‐alkyne cycloaddition. By similar means, metabolic labeling can also be performed with the alkyne‐bearing homopropargylglycine (HPG) and clicked to an azide‐functionalized fluorophore. Curr. Protoc. Cell Biol. 56:7.11.1‐7.11.29. © 2012 by John Wiley & Sons, Inc.

Keywords: FUNCAT; click chemistry; copper(I)‐catalyzed [3+2]azide‐alkyne cycloaddition; AHA; HPG; protein synthesis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Funcat in Cell Lines and Primary Cells
  • Alternate Protocol 1: Funcat in Hippocampal Slices
  • Alternate Protocol 2: Funcat in Larval Zebrafish
  • Alternate Protocol 3: Funcat in Microfluidic Chambers
  • Support Protocol 1: Combination of Funcat with High‐Resolution Fish
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Funcat in Cell Lines and Primary Cells

  Materials
  • Adherent cells from primary cell preparation or cell lines grown on:
    • 18‐mm glass coverslips in a 12‐well culture plate or
    • 12‐mm glass coverslips in a 24‐well culture plate or
    • MatTek glass‐bottom dishes
  • Methionine‐free media [e.g., HBS (see recipe) or methionine‐free DMEM with supplements (see recipe) or methionine‐free Hibernate A with B27 (see recipe)]
  • 100 mM AHA (see recipe)
  • 100 mM methionine (see recipe)
  • 40 mM anisomycin (see recipe)
  • Ice
  • PBS‐MC (see recipe)
  • Fixation solutions: e.g., PFA‐sucrose (see recipe) or PLP (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.4
  • B‐Block (see recipe)
  • PBS‐Tx (see recipe), optional
  • Phosphate‐buffered saline (PBS; see recipe for 10× PBS), pH 7.8
  • 200 mM TBTA in dimethyl sulfoxide (DMSO), triazole ligand (see recipe)
  • 500 mM TCEP in H 2O (see recipe)
  • 2 mM fluorophore‐alkyne‐tag in DMSO (see recipe)
  • 200 mM CuSO 4 in H 2O (see recipe)
  • FUNCAT wash buffer (see recipe)
  • Primary antibody (e.g., rabbit anti‐MAP2 or mouse anti‐MAP2)
  • C‐Block (see recipe), optional
  • Secondary antibody, fluorophore‐coupled (e.g., goat anti‐rabbit‐Alexa 647 or goat anti‐mouse Alexa 647)
  • 1 mg/ml DAPI (see recipe)
  • Mounting medium: e.g., Mowiol or Fluoromount or Aquapolymount
  • Vortex mixer
  • Horizontal shaker
  • FUNCAT incubation plate (see special equipment, Fig. A) or MatTek overhead incubation support (see special equipment, Fig. B)
  • Microscopic slides

Alternate Protocol 1: Funcat in Hippocampal Slices

  Materials
  • Acute 450‐µm hippocampal slices sectioned on a vibratome or tissue slicer (for protocol see Madison and Edson, )
  • Ringer's solution, carbogenated (see recipe)
  • 100 mM AHA (see recipe)
  • 100 mM methionine (see recipe)
  • 40 mM anisomycin (see recipe)
  • PBS‐MC (see recipe)
  • Fixation solutions: e.g., PFA‐sucrose (see recipe) or PLP (see recipe)
  • 3% agarose solution (see recipe)
  • PBS, pH 7.8 (see recipe)
  • Superglue
  • B‐Block (see recipe)
  • PBS‐Tx (see recipe)
  • 200 mM TBTA in dimethyl sulfoxide (DMSO), triazole ligand (see recipe)
  • 500 mM TCEP in H 2O (see recipe)
  • 2 mM fluorophore‐alkyne‐tag in DMSO
  • 200 mM CuSO 4 in H 2O (see recipe)
  • FUNCAT wash buffer (see recipe)
  • PBS‐T, pH 7.4 (see recipe)
  • Primary antibody (e.g., goat anti‐rabbit‐Alexa 647 or goat anti‐mouse Alexa 647)
  • Secondary antibody, fluorophore‐coupled (e.g., goat anti‐rabbit Alexa 647)
  • 1 mg/ml DAPI (see recipe)
  • Mounting medium: e.g., Mowiol or Fluoromount or Aquapolymount
  • Whatman filter paper no. 1
  • 35‐mm tissue culture dishes
  • Interface recovery chamber (Madison and Edson, )
  • Submerged incubation chamber (tissue slice chamber, Harvard Apparatus)
  • Water bath
  • Horizontal shaker
  • Hot plate magnetic stirrer
  • Artist brushes
  • Stereomicroscope
  • Scalpel
  • Forceps
  • Vibratome Leica VT1200S
  • 24‐well plates
  • 15‐ml Falcon tubes
  • Vortex mixer
  • Microscope slides
  • Coverslips

Alternate Protocol 2: Funcat in Larval Zebrafish

  Materials
  • 4 to 6 days post‐fertilization (dpf) larval zebrafish
  • E3 embryo medium (see recipe)
  • 100 mM AHA (see recipe)
  • 5 mg/ml puromycin (see recipe)
  • Ice
  • PFA‐sucrose (see recipe)
  • Methanol
  • PBS‐T (pH 7.4 and 7.8; see recipe)
  • PBDTT (see recipe)
  • Proteinase K (10 µg/ml)
  • Z‐Block (see recipe)
  • 200 mM TBTA in DMSO, triazole ligand (see recipe)
  • 500 mM TCEP in H 2O (see recipe)
  • 2 mM fluorophore‐alkyne‐tag in DMSO (see recipe)
  • 200 mM CuSO 4 in H 2O (see recipe)
  • 0.5 M EDTA
  • Primary antibody
  • Secondary antibody, fluorophore‐coupled
  • 0.6% agarose
  • 50‐mm petri dishes
  • 1.5‐ml microcentrifuge tubes
  • Vortex mixer
  • Rotary shaker
  • MatTek dish
  • Stereomicroscope
  • Microwave

Alternate Protocol 3: Funcat in Microfluidic Chambers

  • Methionine‐free Hibernate A (Brain Bits) with B27 (see recipe)
  • Primary hippocampal neurons cultured in microfluidic chambers (Taylor et al., )
  • 100 mM AHA (see recipe)
  • 100 mM methionine (see recipe)
  • Ice
  • 40 mM anisomycin (see recipe)
  • PBS‐MC (see recipe)
  • Fixation solutions: e.g., PFA‐sucrose (see recipe) or PLP (see recipe)
  • PBS‐Tx (see recipe)
  • C‐Block (see recipe)
  • PBS, pH 7.8 (see recipe)
  • Microfluidic chambers (Taylor et al., ; Fig. C,D; available from Xona Microfluidics LLC: SDN900 or µLP)
  • Syringe pump
  • 37°C incubator
  • Spacer (approximately 3 × 1 × 1–mm3)
  • Humidified chamber

Support Protocol 1: Combination of Funcat with High‐Resolution Fish

  • Cells (see protocol 1 or protocol 4)
  • Affymetrix QG ViewRNA HC Screening Assay kit containing stock solutions for buffers; prepare the following buffers according to the manufacturer's recommendations:
    • Working detergent solution
    • Working protease stop buffer
    • Working probe set diluent
    • RNAView wash buffer
    • Working storage buffer
    • Working amplifier diluent
    • Working label probe diluent
  • Affymetrix QG ViewRNA Probe set for mRNA of interest
  • Affymetrix QG ViewRNA HC Screening Signal Amplification kit (divide into 10‐µl aliquots when thawing for the first time and use aliquots up to three times):
    • Preamplifier
    • Amplifier
    • Label probe
  • Hybridization oven (40°C)
  • Humidified chamber
  • Thermomixer, heating block (40°C)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Agard, N.J., Prescher, J.A., and Bertozzi, C.R. 2004. A strain‐promoted [3 + 2] azide‐alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126:15046‐15047.
   Beatty, K.E. and Tirrell, D.A. 2008. Two‐color labeling of temporally defined protein populations in mammalian cells. Bioorg. Med. Chem. Lett. 18:5995‐5999.
   Beatty, K.E., Fisk, J.D., Smart, B.P., Lu, Y.Y., Szychowski, J., Hangauer, M.J., Baskin, J.M., Bertozzi, C.R., and Tirrell, D.A. 2010. Live‐cell imaging of cellular proteins by a strain‐promoted azide‐alkyne cycloaddition. Chembiochem 11:2092‐2095.
   Beatty, K.E., Szychowski, J., Fisk, J.D., and Tirrell, D.A. 2011. A BODIPY‐cyclooctyne for protein imaging in live cells. Chembiochem 12:2137‐2139.
   Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802‐805.
   Cox, J. and Mann, M. 2011. Quantitative, high‐resolution proteomics for data‐driven systems biology. Annu. Rev. Biochem. 80:273‐299.
   Dieterich, D.C. 2010. Chemical reporters for the illumination of protein and cell dynamics. Curr. Opin. Neurobiol. 20:623‐630.
   Dieterich, D.C. and Link, A.J. 2009. Click chemistry in protein engineering, design, detection and profiling. In Click Chemistry for Biotechnology and Materials Science. (J. Lahann, ed.) pp. 309‐325. John Wiley & Sons, Hoboken, N.J.
   Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A., and Schuman, E.M. 2006. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. U.S.A. 103:9482‐9487.
   Dieterich, D.C., Lee, J.J., Link, A.J., Graumann, J., Tirrell, D.A., and Schuman, E.M. 2007. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non‐canonical amino‐acid tagging. Nat. Protoc. 2:532‐540.
   Dieterich, D.C., Hodas, J.J., Gouzer, G., Shadrin, I.Y., Ngo, J.T., Triller, A., Tirrell, D.A., and Schuman, E.M. 2010. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat. Neurosci. 13:897‐905.
   Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 91:12501‐12504.
   Hinz, F.I., Dieterich, D.C., Tirrell, D.A., and Schuman, E.M. 2012. Noncanonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem. Neurosci. 3:40‐49.
   Hu, Y., Huang, X., Chen, G.Y., and Yao, S.Q. 2004. Recent advances in gel‐based proteome profiling techniques. Mol. Biotechnol. 28:63‐76.
   Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. 1998. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem. 273:23629‐23632.
   Kiick, K.L., Saxon, E., Tirrell, D.A., and Bertozzi, C.R. 2002. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. U.S.A. 99:19‐24.
   Link, A.J., Vink, M.K., and Tirrell, D.A. 2007. Preparation of the functionalizable methionine surrogate azidohomoalanine via copper‐catalyzed diazo transfer. Nat. Protoc. 2:1879‐1883.
   Madison, D. and Edson, E.B. 2001. Preparation of hippocampal brain slices. Curr. Protoc. Neurosci. 00:6.4.1‐6.4.7.
   Ngo, J.T., Champion, J.A., Mahdavi, A., Tanrikulu, I.C., Beatty, K.E., Connor, R.E., Yoo, T.H., Dieterich, D.C., Schuman, E.M., and Tirrell, D.A. 2009. Cell‐selective metabolic labeling of proteins. Nat. Chem. Biol. 5:715‐717.
   Player, A.N., Shen, L.P., Kenny, D., Antao, V.P., and Kolberg, J.A. 2001. Single‐copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49:603‐612.
   Prescher, J.A. and Bertozzi, C.R. 2005. Chemistry in living systems. Nat. Chem. Biol. 1:13‐21.
   Roche, F.K., Marsick, B.M., and Letourneau, P.C. 2009. Protein synthesis in distal axons is not required for growth cone responses to guidance cues. J. Neurosci. 29:638‐652.
   Rostovtsev, V.V., Green, L.G., Fokin, V.V., and Sharpless, K.B. 2002. A stepwise huisgen cycloaddition process: copper(I)‐catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:2596‐2599.
   Taylor, A.M., Dieterich, D.C., Ito, H.T., Kim, S.A., and Schuman, E.M. 2010. Microfluidic local perfusion chambers for the visualization and manipulation of synapses. Neuron 66:57‐68.
   Tcherkezian, J., Brittis, P.A., Thomas, F., Roux, P.P., and Flanagan, J.G. 2010. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell 141:632‐644.
   Tornoe, C.W., Christensen, C., and Meldal, M. 2002. Peptidotriazoles on solid phase: [1,2,3]‐triazoles by regiospecific copper(i)‐catalyzed 1,3‐dipolar cycloadditions of terminal alkynes to azides. J. Org. Chemistry. 67:3057‐3064.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library