Activity‐Based Protein Profiling (ABPP) and Click Chemistry (CC)–ABPP by MudPIT Mass Spectrometry

Anna E. Speers1, Benjamin F. Cravatt1

1 The Skaggs Institute for Chemical Biology and Department of Physiological Chemistry, The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090138
Online Posting Date:  December, 2009
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Abstract

Activity‐based protein profiling (ABPP) is a chemical proteomic method for functional interrogation of enzymes within complex proteomes. This unit presents a protocol for in vitro and in vivo labeling using ABPP and Click Chemistry (CC)‐ABPP probes for in‐depth profiling using the Multi‐dimensional Protein Identification Technology (MudPIT) analysis platform. Curr. Protoc. Chem Biol. 1:29‐41. © 2009 by John Wiley & Sons, Inc.

Keywords: activity‐based protein profiling; ABPP; click chemistry; mass spectrometry; MudPIT; activity‐based probes; biotin; alkyne; azide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Labeling Enzymes In Vitro by ABPP
  • Alternate Protocol 1: Labeling Enzymes in Living Cells or Mice by Click Chemistry–Activity‐Based Protein Profiling (CC‐ABPP)
  • Support Protocol 1: Preparation of Cells/Tissue Homogenates
  • Support Protocol 2: Methanol/Chloroform Precipitation of Proteins
  • Basic Protocol 2: Enrichment and Digestion of Probe‐Labeled Proteins for MudPIT Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling Enzymes In Vitro by ABPP

  Materials
  • 1 mg protein/ml proteome source (e.g., cell or tissue homogenate; see protocol 3)
  • 100× (0.5 to 2 mM) biotinylated ABPP probe stock in dimethylsulfoxide (DMSO) (for details of synthesis, see Evans and Cravatt, ; Cravatt et al., ; and Paulick and Bogyo, ; store up to several years at –20° or –80°C)
  • Dimethylsulfoxide (DMSO)
  • 10% (v/v) Triton X‐100 (store up to several months at room temperature)
  • 50 mM Tris⋅Cl, pH 8.0 (store up to several months at 4°C)
  • 10% (w/v) SDS in water
  • End‐over‐end rotator
  • 10DG disposable chromatography columns (BioRad)
  • 15‐ml conical centrifuge tubes
  • 90°C heating block

Alternate Protocol 1: Labeling Enzymes in Living Cells or Mice by Click Chemistry–Activity‐Based Protein Profiling (CC‐ABPP)

  • Cells growing in culture in 15‐cm plates or laboratory mice
  • 1000× (5 to 25 mM) probe‐alkyne stock in DMSO (for details of synthesis, see Evans and Cravatt, ; Cravatt et al., ; and Paulick and Bogyo, ; store up to several years at −20°C), for cell culture experiment
  • 1× Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen; without calcium and magnesium)
  • 1 to 5 mg/ml probe‐alkyne in vehicle (see recipe)
  • Vehicle
  • 5 mM biotin‐azide (PEG4 carboxamide‐6‐azidohexanyl biotin; Invitrogen) in DMSO (store up to several years at −20°C)
  • 50 mM tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Fluka) in H 2O (prepare fresh prior to use)
  • 1.7 mM TBTA (see recipe)
  • 50 mM CuSO 4⋅5H 2O in H 2O (store up to several months at room temperature; remake if precipitate forms)
  • Methanol, cold
  • 2.5% (w/v) SDS in Ca‐ and Mg‐free D‐PBS (store up to several months at room temperature)
  • Cell scraper
  • Refrigerated centrifuge
  • Animal balance
  • Probe sonicator
  • 60°C water bath or heating block
  • Additional reagents and equipment for preparing cell/tissue homogenates ( protocol 3) and methanol:chloroform precipitation ( protocol 4)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals.

Support Protocol 1: Preparation of Cells/Tissue Homogenates

  Additional Materials
  • Harvested cells/tissue
  • 1× Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen; without calcium and magnesium)
  • Protein assay kit: e.g., DC protein assay (BioRad)
  • Razor blade
  • Glass plate or large tissue culture dish
  • Dounce homogenizer
  • Probe sonicator
  • Refrigerated centrifuge
  • Ultracentrifuge
  • Insulin syringe

Support Protocol 2: Methanol/Chloroform Precipitation of Proteins

  Materials
  • Methanol
  • Chloroform
  • 15‐ml conical centrifuge tubes
  • Refrigerated centrifuge

Basic Protocol 2: Enrichment and Digestion of Probe‐Labeled Proteins for MudPIT Analysis

  Materials
  • SDS‐solubilized protein sample ( protocol 1 or the protocol 2)
  • 1× Dulbecco's phosphate‐buffered saline (D‐PBS; Invitrogen; without calcium and magnesium)
  • Streptavidin beads: 50% (v/v) slurry of Immunopure immobilized streptavidin (Pierce)
  • 1% (w/v) SDS in Ca‐ and Mg‐free D‐PBS (store up to several months at room temperature)
  • 6 M and 2 M urea in Ca‐ and Mg‐free D‐PBS (prepare fresh prior to use)
  • 200 mM dithiothreitol (DTT) in H 2O (prepare fresh daily, or store aliquots at −20°C for months)
  • 500 mM iodoacetamide (IAA) in H 2O (prepare fresh daily, or store aliquots at −20°C for months)
  • 100 mM CaCl 2 in H 2O (store at room temperature for months)
  • 0.5 mg/ml sequence‐grade modified trypsin (Promega) supplied in resuspension buffer
  • 90% formic acid
  • 50% (v/v) acetonitrile/0.1% trifluoroacetic acid (TFA) in H 2O (prepare fresh prior to use)
  • 5% (v/v) formic acid (FA) in H 2O
  • Empty Micro Bio‐Spin Chromatography Columns (BioRad, cat. no. 732‐6204)
  • End‐over‐end rotator
  • Low‐adhesion screw‐top microcentrifuge tubes (Sarstedt)
  • Gel‐loading pipet tips
  • 65°C water bath or heat block
  • SpeedVac evaporator
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Figures

Videos

Literature Cited

Literature Cited
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