Target Identification by Diazirine Photo‐Cross‐Linking and Click Chemistry

Andrew L. MacKinnon1, Jack Taunton2

1 Program in Chemistry and Chemical Biology and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California, 2 Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090167
Online Posting Date:  December, 2009
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Abstract

Target identification is often the rate‐determining step in deciphering the mechanism of action of biologically active small molecules. Photo‐affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine‐based photo‐affinity probes and Cu(I)‐catalyzed click chemistry to covalently label and visualize the targets of biologically active small molecules. A general method for affinity purification of probe‐modified proteins, useful for identification of protein targets, is also described. Curr. Protoc. Chem Biol. 1:55‐73. © 2009 by John Wiley & Sons, Inc.

Keywords: photo‐affinity labeling; diazirine; click chemistry; target identification; affinity purification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Diazirine Photoactivation and Cu(I)‐Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets
  • Support Protocol 1: Affinity Purification of Probe‐Modified Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Diazirine Photoactivation and Cu(I)‐Catalyzed Click Chemistry for Covalent Labeling and Detection of Protein Targets

  Materials
  • Endoplasmic reticulum (ER) microsomes (∼1 mg/ml total protein) or other soluble or membrane protein lysate containing the unknown macromolecular target, in PBS (see recipe for PBS)
  • 0.8 mM stock solution of photostable competitor compound (labeled 3 in Fig. )
  • Dimethylsulfoxide (DMSO)
  • 20 µM stock solution of photo‐affinity probe (labeled 2 in Fig. ) in DMSO
  • 10% (w/v) sodium dodecyl sulfate (SDS) in H 2O
  • 5 mM TAMRA‐azide (labeled 4 in Fig. ) or biotin‐azide (labeled 5 in Fig. ), synthesized by published methods (Speers and Cravatt, ; Weerapana et al., ); similar reagents are available commercially from Invitrogen, e.g., PEG4 carboxamide‐6‐azidohexanyl biotin (Fig. )
  • 1.7 mM TBTA in 80% t‐butanol/20% DMSO (see recipe)
  • 50 mM CuSO 4 in H 2O
  • 50 mM Tris(2‐carboxyethyl)phosphine (TCEP) in H 2O, adjusted to pH ∼7 with 1 M NaOH; prepare immediately before use
  • 6× Laemmli sample buffer (see recipe)
  • Fluorescent molecular weight markers (Pierce)
  • 96‐well plate or other open, shallow container
  • 1000 W Hg(Xe) lamp (Oriel Instruments, model 66923) with band‐pass filter for irradiation at ∼355 nm (Oriel Instruments, cat. no. 59810) and a filter to absorb heat (Oriel Instruments, cat. no. 59044); http://www.oriel.com/
  • 0.5‐ml polypropylene microcentrifuge tubes
  • Typhoon 9400 phosphor imager (Amersham)
  • Additional reagents and equipment for SDS‐PAGE (e.g., Gallagher, ) and immunoblotting (western blotting ; e.g., Gallagher et al., )

Support Protocol 1: Affinity Purification of Probe‐Modified Proteins

  • Protein mixture labeled with photo‐affinity probe ( protocol 1, steps 1 to 5)
  • Liquid N 2
  • Acetone cooled to −20°C
  • 1% SDS in PBS (see recipe for PBS)
  • Affinity purification buffer (see recipe)
  • Protein A–Sepharose beads (GE Healthcare)
  • Anti‐TAMRA antibody (Invitrogen, cat. no. A6397)
  • Monomeric avidin–agarose beads (Pierce)
  • Wash buffer (see recipe)
  • Elution buffer (see recipe)
  • Polyallomer 1.5‐ml microcentrifuge tubes (Beckman‐Coulter)
  • Benchtop ultracentrifuge
  • Sonicating water bath
  • Refrigerated microcentrifuge
  • Rotating tube mixer
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Figures

Videos

Literature Cited

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Key References
   Best, M.D. 2009. See above.
  A recent review of bio‐orthogonal click chemistry methods.
   Brunner, 1993. See above.
  An excellent introduction to the structure and chemistry of photoreactive groups and their use in photo‐affinity labeling in biological systems.
   Colca et al., 2004. See above.
  An excellent example of PAL for identifying a novel integral membrane target of a therapeutically relevant small molecule.
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