Bioorthogonal Chemical Reporters for Analyzing Protein Sulfenylation in Cells

Thu H. Truong1, Kate S. Carroll2

1 Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 2 Department of Chemistry, The Scripps Research Institute, Jupiter, Florida
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110219
Online Posting Date:  June, 2012
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Abstract

Protein sulfenylation (RSOH), the redox‐based modification of cysteine thiol side chains by hydrogen peroxide (H2O2), is an important mechanism in signal transduction. Likewise, dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments. To address this problem, selective chemical reporters have been developed for the detection and identification of sulfenylated proteins directly in cells. In the approach described here, a cyclic β‐diketone warhead is functionalized with an azide or alkyne chemical handle. An orthogonally functionalized biotin or fluorescent reporter is then appended to the probe post‐homogenization via click chemistry for downstream analysis. These bi‐functional probes are exquisitely selective for protein sulfenyl modifications, non‐toxic, and do not perturb intracellular redox balance. These reagents have been utilized to investigate sulfenylation in vitro and to identify intracellular protein targets of H2O2 during cell signaling. These methods provide a facile way to detect protein sulfenic acids and to study the biological role of cysteine oxidation with regard to physiological and pathological events. Curr. Protoc. Chem. Biol. 4:101‐122 © 2012 by John Wiley & Sons, Inc.

Keywords: thiol modification; protein sulfenylation; redox signaling; click chemistry; in‐gel fluorescence; western blotting

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Labeling Sulfenylated Proteins In Vitro
  • Basic Protocol 2: Labeling Endogenous Sulfenylated Proteins in Cell Suspension
  • Basic Protocol 3: Labeling Exogenous Sulfenylated Proteins in Cell Suspension
  • Basic Protocol 4: Immunoblot Detection of Biotinylated Proteins
  • Basic Protocol 5: In‐Gel Detection of Fluorophore‐Tagged Proteins
  • Support Protocol 1: Pre‐Clearing Cell Lysates of Endogenous Biotinylated Proteins
  • Support Protocol 2: Methanol Precipitation of Proteins
  • Support Protocol 3: Methanol/Chloroform Precipitation of Proteins
  • Alternate Protocol 1: On‐Plate Labeling of Endogenous Protein Sulfenylation in Cells
  • Alternate Protocol 2: On‐Plate Labeling of Exogenous Protein Sulfenylation in Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling Sulfenylated Proteins In Vitro

 Materials
  • GAPDH, lyophilized powder (Sigma‐Aldrich) or other purified protein of interest
  • Tris labeling buffer (see recipe)
  • 50 mM tris(2‐carboxyethyl) phosphine hydrochloride (TCEP; Sigma‐Aldrich), prepared fresh in water
  • Bio‐Spin 6 columns, pre‐packed in Tris buffer (BioRad)
  • DMSO (vehicle; Sigma‐Aldrich)
  • 25 mM DAz‐2 (Cayman Chemicals) or DYn‐2 (Cayman Chemicals), prepared in DMSO
  • 1 mM H2O2 stock (Sigma‐Aldrich), prepared fresh in water and maintained on ice
  • Click labeling buffer (see recipe)
  • 5 mM biotin tag (biotin alkyne or azide; Invitrogen) in DMSO or 5 mM fluorescent tag (TAMRA or AlexaFluor488 azide; Invitrogen) in DMSO
  • 2 mM tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl] amine (TBTA; Sigma Aldrich), prepared in 4:1 DMSO/t‐butanol (TBTA can also be synthesized by published methods; Chan et al., 2004)
  • 50 mM CuSO4, prepared fresh in water
  • 1× PBS (Boston BioProducts)
  • 2× Laemmli sample buffer with 10% β‐mercaptoethanol (BioRad)
  • Mini‐Protean TGX 4% to 15% Tris‐Glycine protein gels (BioRad)
  • Centrifuge
  • NanoDrop2000c spectrophotometer (Thermo Scientific)
  • 37°C incubator with shaker
  • Platform shaker
  • 95°C heating block

Basic Protocol 2: Labeling Endogenous Sulfenylated Proteins in Cell Suspension

 Materials
  • A431 cells (ATCC)
  • DMEM complete culture medium (high‐glucose DMEM supplemented with 10% FBS, 1% GlutaMax, 1% MEM nonessential amino acids, and 1% penicillin‐streptomycin; Invitrogen)
  • 1× PBS (Boston BioProducts)
  • DMEM only (serum‐free, high‐glucose DMEM; Invitrogen)
  • 30 µg/ml EGF stock (BD Biosciences), prepared in H2O and kept on ice
  • 0.25% trypsin (Invitrogen)
  • 250 mM DAz‐2 (Cayman Chemicals) or DYn‐2 (Cayman Chemicals), prepared in DMSO
  • DMSO (vehicle; Sigma‐Aldrich)
  • Modified RIPA lysis buffer supplemented with EDTA‐free protease inhibitors and 200 U/ml catalase (see recipe)
  • BCA protein assay (Pierce)
  • 5 mM biotin tag (biotin alkyne or azide; Invitrogen) in DMSO or 5 mM fluorescent tag (TAMRA or AlexaFluor488 azide; Invitrogen) in DMSO
  • 50 mM TCEP (Sigma‐Aldrich), prepared fresh in water
  • 2 mM TBTA (Sigma Aldrich), prepared in 4:1 DMSO/t‐butanol (TBTA also synthesized by published methods; Chan et al., 2004)
  • 50 mM CuSO4, prepared fresh in water
  • 0.5 M EDTA (Boston BioProducts)
  • 10% SDS, prepared in H2O
  • 2× Laemmli sample buffer with 10% β‐mercaptoethanol (BioRad)
  • Mini‐Protean TGX 4% to 15% Tris‐Glycine protein gels (BioRad)
  • 37°C incubator
  • Refrigerated centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 95°C heating block
  • Vortex

Basic Protocol 3: Labeling Exogenous Sulfenylated Proteins in Cell Suspension

 Materials
  • HepG2 cells (ATCC)
  • MEM complete culture medium (MEM supplemented with 10% FBS, 1% GlutaMax, 1% MEM nonessential amino acids, and 1% penicillin‐streptomycin; Invitrogen)
  • 1× PBS (Boston BioProducts)
  • MEM with 0.5% FBS (MEM supplemented with 0.5% FBS; Invitrogen)
  • 100 mM H2O2 stock (Sigma‐Aldrich), prepared fresh in water and maintained on ice
  • Additional reagents and equipment (see Basic Protocol 2)

Basic Protocol 4: Immunoblot Detection of Biotinylated Proteins

 Materials
  • SDS‐PAGE gel with resolved samples
  • PVDF membrane (0.2‐µm; BioRad)
  • 3% BSA (Fisher), prepared in TBST
  • TBST (Boston BioProducts)
  • Streptavidin‐HRP antibody (GE‐Healthcare)
  • ECL Plus western blot detection system (GE Healthcare)
  • GAPDH antibody (Santa Cruz Biotechnology)
  • Rabbit anti‐mouse IgG‐HRP (Invitrogen)
  • X‐ray film

Basic Protocol 5: In‐Gel Detection of Fluorophore‐Tagged Proteins

 Materials
  • SDS‐PAGE gel with resolved samples
  • Destain solution (see recipe)
  • SYPRO ruby protein stain (BioRad)
  • Wash solution (see recipe)
  • Platform rocker
  • Fluorescence gel scanner (e.g., Amersham Biosciences Typhoon 9400 variable mode imager)

Support Protocol 1: Pre‐Clearing Cell Lysates of Endogenous Biotinylated Proteins

 Materials
  • NeutrAvidin agarose resin (Pierce)
  • Modified RIPA buffer (see recipe)
  • Cell lysates
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge
  • Platform rocker at 4°C

Support Protocol 2: Methanol Precipitation of Proteins

 Materials
  • Click chemistry reaction of cell lysate
  • Methanol, ice‐cold
  • Refrigerated centrifuge

Support Protocol 3: Methanol/Chloroform Precipitation of Proteins

 Materials
  • Click chemistry reaction of cell lysate
  • Methanol, ice‐cold
  • Chloroform, ice‐cold
  • Refrigerated centrifuge
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Figures

  •  FigureFigure 1. Bioorthogonal detection of protein cysteine oxidation. (A) Oxidative modifications of protein cysteines. Low pKa thiols susceptible to oxidation can react with H2O2 to form a sulfenic acid, also known as sulfenylation. This modification may be stabilized by the protein microenvironment or condense with a second thiol to form an intra‐ or inter‐molecular disulfide. Alternatively, the sulfenic acid can undergo further oxidation to the sulfinic or sulfonic acid under conditions of high oxidative stress, typically associated with disease states. (B) Chemoselective reaction between sulfenic acid and 5,5‐dimethyl‐1,3‐cyclohexanedione (dimedone, 1). (C) Selective probes for detecting protein sulfenic acids based on the 1,3‐cyclohexanedione scaffold (2). These probes are functionalized with azide (3‐5) or alkyne (6‐8) chemical reporter groups, allow for relative quantification of sulfenic acids (9‐10), and can target specific classes of redox‐regulated proteins, such as protein tyrosine phosphatases (PTPs) (11‐12).
    View Image
  •  FigureFigure 2. Cell‐based detection of protein sulfenylation. (A) Strategy to detect protein sulfenylation in living cells. Sulfenic acids are labeled in situ using selective, cell‐permeable chemical probes. Cells are then washed, homogenized, and probe‐labeled proteins are conjugated to a biotin or fluorescent tag via the bioorthogonal click chemistry reaction. This approach enables downstream detection by immunoblot or in‐gel fluorescence. Alternatively, biotinylated proteins may be enriched for proteomic analysis. (B) Generic scheme for click chemistry bioconjugation. (C) Biotin tags utilized in this study. (D) Fluorescent tags utilized in this study.
    View Image
  •  FigureFigure 3. Detection of sulfenyl modifications with purified protein in vitro. GAPDH was stimulated with H2O2 and labeled with DAz‐2 or DYn‐2 for 1 hr. Probe‐labeled GAPDH can be detected by streptavidin‐HRP immunoblot (A‐B) or in‐gel fluorescence (C‐D). Equal protein loading is demonstrated by reprobing the immunoblot with antibodies to GAPDH (immunoblot) or by SYPRO red dye staining of the SDS‐PAGE gel (fluorescence).
    View Image
  •  FigureFigure 4. Detection of EGF‐mediated protein sulfenylation in A431 cells. A431 cells were stimulated with 100 ng/ml EGF for 5 min. EGFR activation leads to the production of endogenous H2O2 and concomitant changes in protein sulfenylation. Cells were labeled with DAz‐2 or DYn‐2 for 1 hr. Sulfenylated proteins can be detected by streptavidin‐HRP immunoblot (A‐B) or in‐gel fluorescence (C‐D). Equal protein loading is demonstrated by reprobing the immunoblot with antibodies to GAPDH (immunoblot) or by SYPRO red dye staining of the SDS‐PAGE gel (fluorescence).
    View Image
  •  FigureFigure 5. Global changes of protein sulfenylation in HepG2 cells exposed to exogenous H2O2. HepG2 cells were treated with 500 µM H2O2 for 5 min. Cells were labeled with DAz‐2 or DYn‐2 for 1 hr. Sulfenylated proteins can be detected by streptavidin‐HRP immunoblot (A‐B) or in‐gel fluorescence (not shown). Equal protein loading is demonstrated by reprobing the immunoblot with antibodies to GAPDH (immunoblot).
    View Image
  •  FigureFigure 6. Labeling and detection of protein sulfenylation in adherent A431 and HepG2 cells. Cells were exposed to 100 ng/ml EGF (A431) or 500 µM H2O2 (HepG2) for 5 min. After treatment, cells remained attached to the tissue culture plate and were labeled with DAz‐2 for the indicated times. Sulfenylated proteins were visualized by streptavidin‐HRP immunoblot for (A) A431 cells and (B) HepG2 cells. Equal protein loading is demonstrated by reprobing the immunoblot with antibodies to GAPDH (immunoblot).
    View Image

Videos

Literature Cited

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