Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins

Abstract

Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.

Figure 4.1

Reaction of DCP-Bio1 with cysteine sulfenic acid. The sulfenic acid-reactive reagent designated DCP-Bio1 [3-(2,4-dioxocyclohexyl)propyl 5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate] is shown in its enol form, reacting with a protein sulfenic acid to generate a stable, alkylated form of the protein (Poole et al., 2007).

Figure 4.2

Use of various elution conditions to recover biotinylated proteins from streptavidin beads. A. Biotinylated AhpC (180 μg) was incubated with 80 μl streptavidin beads in 250 μl total volume of 2 M urea in PBS, pH 7.2 and incubated for 1.5 h at 24 °C. Beads were washed 3 ×0.5 ml 2 M urea in PBS, pH 7.2 and aliquotted into five different tubes prior to centrifugation for 2 min at 1000×g and removal of the supernatant. Elution solutions (10 μl) were added to each tube and incubated at the appropriate temperature for 10 min. Elution conditions were: 2% SDS in 50 mM Tris–HCl, 1 mM EDTA, pH 8.0 (90 °C); 50 mM Tris–HCl, 1 mM EDTA, pH 8.0 (90 °C); 8 M urea with 2% CHAPS (37 °C); 8 M guanidine hydrochloride (GuHCl) (37 °C); and 100 mM glycine, pH 2.8 (24 °C). The amount of AhpC protein in 2 μl of the eluted fraction was visualized by Coomassie Blue stain after SDS–PAGE. B. Sulfenic acid-containing C165S AhpC labeled with DCP-Bio1 was incubated with monoavidin beads overnight, washed three times with PBS buffer, and aliquotted into separate tubes. Samples were either incubated with 2.25 M ammonium hydroxide for 16 h at 24 °C or boiled in SDS sample buffer containing β-mercaptoethanol for 10 min, and AhpC in the eluant was visualized as in (A).

Figure 4.3

Time course of sulfenic acid formation in individual proteins monitored by Western blot analysis. HEK293 cells were stimulated with TNFα for the indicated amount of time, cells were washed with PBS, and sulfenic acids were trapped by the addition of lysis buffer containing 1 mM DCP-Bio1, 10 mM N-ethylmaleimide, 10 mM iodoacetamide, 200 U/ml catalase, 100 mM NaCl, 100 μM DTPA, 20 mM β-glycerophosphate, 1 mM sodium vanadate, 50 mM sodium fluoride, 1 mM PMSF, 0.01 mg/ml aprotinin, 0.01 mg/ml leupeptin, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxy-cholate, 0.5% NP-40, and 0.5% Triton-X-100 in 50 mM Tris–HCl, pH 7.5, incubated on ice for 1 h, and stored at −80 °C. Biotinylated proteins were affinity captured according to the protocol described in methods, then separated by SDS–PAGE and transferred to a nitrocellulose membrane. The extent of cysteine oxidation on individual proteins was evaluated using protein-specific antibodies. Biotin-labeled AhpC was added based on protein concentrations prior to affinity capture and used as a procedural control for the biotin-based affinity capture, elution and gel loading steps. Antibodies to HSP70, PKC-β1, and GAPDH were from Santa Cruz; antibodies to PTEN and SHP-2 were from Cell Signaling, and the AhpC antibody was purified from rabbit serum.

Figure 4.4

SHP-2 (A) and PTEN (B) labeling is established by immunoblots to SHP-2 and PTEN after biotin affinity capture (biotin AC) and by visualizing biotinylated proteins after immunoprecipitation (IP) of SHP-2 and PTEN, but these proteins are not endogenously biotinylated. HEK293 cells were grown in DMEM media containing 10% FBS and exchanged into serum-free DMEM media for 30 min prior to the addition of the 5 mM DCP-Bio1-containing lysis buffer and incubation (see Fig. 4.3). Cell lysates were centrifuged at 14,000×g for 10 min, and the protein concentration in each supernatant was determined using the BCA protein assay. For immunoprecipitation, lysates (175 μg of protein each) were preincubated for 1 h at 4 °C with 20 μl Dynabeads-Protein A magnetic beads preequilibrated in PBS (but lacking antibody) to remove nonspecifically associating proteins, then supernatants were transferred to tubes containing 0.5 μl of either anti-PTEN or anti-SHP-2 antibody (Cell Signaling) and a fresh aliquot (20 μl) of magnetic beads and incubated overnight at 4 °C. Supernatants were then removed and the beads were washed three times with PBS buffer. Immunoprecipitated proteins were eluted with 35 μl SDS sample buffer. Affinity capture of biotiny-lated proteins was conducted according to the protocol described in methods. Proteins (10 μl per sample) were separated by SDS–PAGE in 10% polyacrylamide gels, transferred to nitrocellulose membrane, and blocked with 5% milk. For immunoblotting, membranes were incubated for 2 h at 24 °C with a 1:10,000 dilution of streptavidin–HRP or for 16 h at 4 °C with a 1:1000 dilution of either anti-SHP-2 (A, Cell Signaling) or anti-PTEN antibodies (B, Cell Signaling), then visualized with Pico chemiluminescent substrate (Pierce).

Figure 4.5

Techniques to monitor global sulfenic acid formation. (A) To visualize protein bands in samples after affinity capture of biotinylated proteins, HEK293 cells were treated (or not) with 100 nM insulin for 2 min, then sulfenic acids were trapped with 1 mM DCP-Bio1 in lysis buffer as in Fig. 4.3. Streptavidin–agarose beads were used to capture biotinylated proteins from lysates, then extensively washed with 1% SDS, 4M urea in PBS, 1 M NaCl, 100 μM ammonium bicarbonate and dH₂O. Proteins were eluted with 2% SDS in 50 mM Tris–HCl, pH 8.0, 1 mM EDTA, separated by SDS–PAGE, and stained with SYPRO Ruby (Pierce). Proteins showing increased sulfenic acid labeling in response to insulin are marked with arrows. (B) To blot for biotin after separation of total protein samples on gels, HEK293 cells were treated (or not) with TNFα for the indicated amount of time, then sulfenic acids were trapped with 1 mM DCP-Bio1 in lysis buffer. Unreacted DCP-Bio1 was removed using a Bio-Gel P6 spin column and 20 μg of protein from each sample was separated on a 10% SDS-PAGE followed by transfer to nitrocellulose. Membranes were blocked overnight with 5% milk, incubated with a 1:1000 dilution of antibiotin, HRP-conjugated antibody (Cell Signaling) for 2 h at 24 °C, and visualized with Pico chemiluminescence kit. As a loading control, 10 μg of each sample was probed for actin using an anti-actin antibody (Cell Signaling). (C) For protein analyses using 2D gels, HEK293 cells were treated with TNFα or insulin and labeled with 1 mM DCP-FL1. Proteins were precipitated with cold acetone, washed with 10% TCA and 1:1 ether:ethanol, and then resolubilized in 8 M urea, 2% CHAPS, 50 mM DTT, and 0.2% ampholytes. Proteins were separated by 2D electrophoresis using pH 3–10 IPG strips (BioRad), followed by 4–20% Criterion gel (BioRad), and fluorescein labeled proteins were visualized on a Amersham STORM 840 fluorescence imager. (D) A FluoReporter biotin quantitation assay kit (Invitrogen) was used to quantify the amount of biotin incorporation into HEK293 cells before and after stimulation with TNFα for 10 min. Briefly, cell lysates (~10–25 μg) were digested with Streptomyces griseus Protease type XIV overnight at 37 °C and diluted in PBS until each sample contained 1–2 μg total protein. After incubation for 5 min at room temperature in the dark with an equal volume of Biotective Green reagent, fluorescence was measured on a Tecan Safire 2 fluorescence plate reader using λ_ex = 485 nm and λ_em = 530 nm.

Figure 4.6

LC/MS/MS spectrum of a DCP-Bio1-labeled tryptic peptide from PrxVI labeled on the reactive Cys residue, Cys46. HEK293 cells were grown in DMEM media to 80% confluence, serum-starved for 20 h, and treated with 100 nM TPA for 1 min. Cells were scraped, then lysed in lysis buffer containing 5 mM DCP-Bio1 and 10 mM NEM and incubated at room temperature for 30 min; proteins in the sample were further treated under denaturing conditions by adding urea to 6M and incubating for 10 min with 10 mM DTT, then adding NEM to 20 mM for another 35 min incubation. To remove excess labeling agents and protease inhibitors, the sample was exchanged into digestion buffer (2 M urea, 100 mM ammonium bicarbonate) using a G-25 spin column. Trypsin was added (1:100, w/w) and incubated at 37 °C for 15 h. Excess trypsin and undigested protein was removed by passing peptides through a 5000 molecular weight cut-off Ultrafree-MC centrifugal filter (Millipore) following digestion. For biotin affinity capture, peptides were incubated with a preequilibrated mono-avidin resin (from Pierce) at 4 °C overnight. The beads were washed several times with additional urea/bicarbonate buffer, followed by 10 mM ammonium bicarbonate, then water. Peptides were eluted with 30% acetonitrile containing 500 mM formic acid, then concentrated in a SpeedVac before analysis. Reverse phase chromatography on a C-18 column was used to resolve peptides, with a portion of the eluant injected directly into the LTQ mass spectrometer. As illustrated above, cleavage of the amide bond results in N-terminal fragments designated as “b” and C-terminal fragments designated as “y”. The masses of both sets of ions are consistent with DCP-Bio1 linked covalently to Cys46 of human peroxiredoxin VI (PrxVI) (y7 – y6 = b6 – b5 = 498.5 m/z).