Visualization and Identification of Fatty Acylated Proteins Using Chemical Reporters

Jacob S. Yount1, Mingzi M. Zhang1, Howard C. Hang1

1 The Rockefeller University, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100225
Online Posting Date:  May, 2011
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Abstract

Protein fatty acylation, the covalent addition of a lipid chain at specific amino acids, changes the inherent hydrophobicity of a protein, often targeting it to cellular membrane compartments and regulating protein activity, stability, and interactions. Fatty acylation can be analyzed using chemical reporters that mimic natural lipids and contain bioorthogonal chemical handles, allowing them to be reacted with detection tags such as fluorophores or affinity tags. Our laboratory has used alkynyl‐chemical reporters of protein myristoylation, S‐palmitoylation, prenylation, and acetylation to provide robust, nonradioactive methods for examining the acylation states of full cellular proteomes and individual proteins of interest by (1) metabolically incorporating these chemical reporters into proteins in living cells; (2) selectively reacting the labeled proteins in cell lysates with azido‐rhodamine via click chemistry, and globally visualizing them with fluorescence gel scanning; (3) analyzing protein acylation on individual candidate proteins using immunoprecipitation, click chemistry, and fluorescence gel scanning; and (4) identifying novel fatty acylated proteins by reacting chemical reporter–labeled proteins with azido‐biotin via click chemistry and selective retrieval using streptavidin beads. This is particularly valuable for the examining protein S‐palmitoylation, which does not occur in readily predicted consensus amino acid motifs. Curr. Protoc. Chem. Biol. 3:81‐97 © 2011 by John Wiley & Sons, Inc.

Keywords: fatty acylation; S‐palmitoylation; click chemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Metabolic Incorporation of Chemical Reporters of Protein Fatty Acylation in Living Cells
  • Basic Protocol 2: Global Fluorescent Profiling of Fatty Acylated Proteins in Whole‐Cell Lysates
  • Basic Protocol 3: Fluorescent Visualization of Fatty Acylation on Candidate Proteins
  • Basic Protocol 4: Proteomic Identification of Proteins Labeled with Chemical Reporters of Fatty Acylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Metabolic Incorporation of Chemical Reporters of Protein Fatty Acylation in Living Cells

  Materials
  • Cultured cells of interest, growing in appropriate complete cell culture medium
  • Serum‐free cell culture medium appropriate for growing the cell type of interest, supplemented with 2% (v/v) charcoal/dextran‐stripped fetal bovine serum (FBS; e.g., Invitrogen), 37°C
  • DMSO (USB grade)
  • 50 mM alkyne‐fatty acid chemical reporter stock in DMSO, e.g.,
    • Alk‐12 (Charron et al., ; for studying myristoylation)
    • Alk‐16 (Sigma‐Aldrich cat no. O8382; also see Charron et al., ; for studying palmitoylation)
    • Alk‐FOH (Charron et al., ; for studying prenylation)
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • Liquid nitrogen or dry‐ice/ethanol bath
  • 37°C, 5% CO 2 cell culture incubator
  • Cell scrapers
  • Refrigerated centrifuge

Basic Protocol 2: Global Fluorescent Profiling of Fatty Acylated Proteins in Whole‐Cell Lysates

  Materials
  • Chemical reporter–labeled cells ( protocol 1), e.g., ∼1 × 106 HeLa cells/pellet
  • 4% (w/v) sodium dodecyl sulfate (SDS) with EDTA‐free protease inhibitors (see recipe)
  • 250 U/µl Benzonase (Sigma‐Aldrich, ultrapure)
  • BCA assay reagents (Pierce Protein Research)
  • 5 mM azido‐rhodamine(Charron et al., ) in dimethyl sulfoxide (DMSO) or tetramethylrhodamine‐5‐carbonyl azide (Invitrogen; see Martin and Cravatt, ) in DMSO
  • 50 mM tris(2‐carboxyethyl)phosphine (TCEP): prepare fresh
  • 2 mM tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl] amine (TBTA) in 1:4 (v/v) DMSO/butanol
  • 50 mM CuSO 4: prepare fresh
  • Methanol, ice‐cold
  • Chloroform, ice‐cold
  • Water, ice‐cold
  • 4× loading buffer (see recipe)
  • 2‐mercaptoethanol (2‐ME)
  • 18‐well 4% to 20% Tris⋅Cl protein gels (Bio‐Rad)
  • Fluorescent protein molecular weight ladder (15 to 250 kDa; Bio‐Rad), diluted as necessary
  • Fluorescence gel destaining solution: 40% (v/v) methanol/50% (v/v) acetic acid/10% (v/v) water
  • Coomassie blue staining reagents (Pierce Protein Research)
  • 1.5‐ml microcentrifuge tubes
  • Bath sonicator (optional)
  • 95°C heating block
  • Fluorescence gel scanner with a 532‐nm excitation and 580‐nm detection filters and 30‐nm band‐pass (e.g., Typhoon 9400, Amersham Biosciences)
  • Additional reagents and equipment for quantifying protein concentration using BCA (Olson and Markwell, ), carrying out SDS‐PAGE (Gallagher, ), and staining proteins (e.g., see Sasse and Gallagher, )

Basic Protocol 3: Fluorescent Visualization of Fatty Acylation on Candidate Proteins

  Materials
  • Chemical reporter–labeled cells ( protocol 1), e.g., ∼2 × 106 HeLa cells/pellet
  • 1% (w/v) Brij97 buffer with EDTA‐free protease inhibitors (see recipe)
  • BCA assay reagents (Pierce Protein Research)
  • 4% (w/v) SDS buffer with EDTA‐free protease inhibitors (see recipe)
  • 5 mM azido‐rhodamine (Charron et al., ) in DMSO or tetramethylrhodamine‐5‐carbonyl azide (Invitrogen; see Martin and Cravatt, ) in DMSO
  • 50 mM tris(2‐carboxyethyl)phosphine (TCEP): prepare fresh
  • 2 mM tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl] amine (TBTA) in 1:4 (v/v) dimethyl sulfoxide (DMSO)/butanol
  • 50 mM CuSO 4: prepare fresh
  • 4× loading buffer (see recipe)
  • 2‐mercaptoethanol (2‐ME)
  • 25% (w/v) hydroxylamine (NH 2OH), pH 7.0 (optional)
  • Fluorescent protein molecular weight ladder (10 to 250 kDa, dual color; Bio‐Rad), diluted as necessary
  • 18‐well 4% to 20% Tris⋅Cl protein gels (Bio‐Rad)
  • Fluorescence gel destaining solution: 50% (v/v) methanol/10% (v/v) acetic acid/40% (v/v) H 2O
  • 1.5‐ml microcentrifuge tubes
  • 95°C heating block
  • Fluorescence gel scanner with a 532‐nm excitation and 580 nm detection filter and 30 nm band‐pass (e.g., Typhoon Amersham Biosciences 9400, scanner)
  • Additional reagents and equipment for quantifying protein concentration using BCA (Olson and Markwell, ), performing immunoprecipitation techniques (Bonifacino et al., ), carrying out SDS‐PAGE (Gallagher, ), staining proteins on gels (Sasse and Gallagher, ), and performing immunoblotting (Gallagher et al., )

Basic Protocol 4: Proteomic Identification of Proteins Labeled with Chemical Reporters of Fatty Acylation

  Materials
  • Chemical reporter–labeled cells ( protocol 1), e.g., ∼20 × 106 HeLa cells/pellet
  • 4% (w/v) SDS buffer with EDTA‐free protease inhibitors (see recipe)
  • 250 U/µl Benzonase
  • BCA assay reagents (Pierce Protein Research)
  • 5 mM azido‐azo‐biotin (Yang et al., ) or azide‐biotin (Invitrogen; also see Martin and Cravatt, )
  • 50 mM and 200 mM tris(2‐carboxyethyl)phosphine (TCEP): prepare fresh
  • 2 mM tris[(1‐benzyl‐1H ‐1,2,3‐triazol‐4‐yl)methyl] amine (TBTA) in 1:4 (v/v) dimethyl sulfoxide (DMSO)/butanol
  • 50 mM CuSO 4: prepare fresh
  • Methanol, ice‐cold
  • Chloroform, ice‐cold
  • Water, ice‐cold
  • 0.5 M EDTA
  • 1% Brij97 buffer with EDTA‐free protease inhibitors (see recipe)
  • Streptavidin agarose beads (Thermo Scientific)
  • PBS, ice cold, pH 7.4 (see recipe)
  • PBS/0.2% (w/v) SDS, pH 7.4 (ice‐cold)
  • ABC buffer (250 mM ammonium bicarbonate), ice‐cold
  • 8 M urea
  • 400 mM iodoacetamide: prepare fresh
  • 1% (w/v) SDS Na 2S 2O 4 elution buffer (see recipe)
  • NuPAGE 4× LDS buffer (Invitrogen): dilute to 1× before use)
  • 2‐mercaptoethanol (2‐ME)
  • Fluorescent protein molecular weight ladder (15 to 250 kDa; Bio‐Rad)
  • 18‐well 4% to 20% Tris⋅Cl protein gels (Bio‐Rad)
  • Coomassie blue staining reagents (Pierce Protein Research)
  • 15‐ml conical, polypropylene tubes
  • Nutating mixer
  • 2‐ml dolphin microcentrifuge tubes
  • Green Centricon devices (YM‐10 membranes with 10 kDa molecular weight cutoff; Millipore)
  • Rotary evaporater (e.g., SpeedVac)
  • 95°C heating block
  • Additional reagents and equipment for quantifying protein concentration using BCA (Olson and Markwell, ), carrying out SDS‐PAGE (Gallagher, ), and staining gels for protein (Sasse and Gallagher, )
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Figures

Videos

Literature Cited

Literature Cited
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