In Vivo Tagging and Characterization of S-Glutathionylated Proteins by a Chemoenzymatic Method

Glutathione (GSH), a sulfhydryl-containing tripeptide present in most organisms at millimolar levels, plays a crucial role in redox homeostasis.1 Reactive cysteine residues are vulnerable to reactive oxygen or nitrogen species and thus depend heavily on GSH to avoid irreversible oxidation.[1a], [2] Reversible conjugation of GSH to proteins through the formation of mixed disulfide bonds is termed protein glutathionylation (PSSG), which additionally alters or regulates protein functions in biological processes, including energy metabolism, signal transduction, ion transport, cytoskeletal assembly, and protein folding.[2a] Although various possible mechanisms have been proposed for PSSG,[1a] the delineation of its functional consequences in vivo remains a longstanding challenge owing to lack of appropriate tools to globally identify this important modification with high sensitivity.[3]

Supporting Information Figure 1. The reactions catalyzed by E. coli glutathionylspermidine synthetase/amidase. Glutathionylspermidine synthetase/amidase is a bifunctional enzyme with an amidase domain at the N terminus and a C-terminal synthetase domain. Biotin-spm and GSH are docked into the active site of GspS (PDB ID:2IOA). The model was created using PyMOL. The carbon, oxygen and nitrogens atoms of biotin-spm and GSH are represented in green, red and blue, respectively. Protein carbon, oxygen, and nitrogen atoms, are shown in grey, red and blue, respectively. Figure 3. A plot of V 0 versus biotin-spm concentration to determine K m and k cat values Reaction rate (V 0 ) was plotted against the concentration of biotin-spm. To confirm whether biotin-spm is a substrate for Gsp synthetase, recombinant Gsp synthetase were incubated with various concentrations of biotin-spm and the resulting activities were assessed by measuring the consumption of NADH by the pyruvate kinase/lactate dehydrogenase-coupled assay. The K m and k cat values of biotin-spm were determined by a previously reported spectrometric assay and found to be similar to those of the native substrate spermidine (K m = 76 μM and k cat = 4.6 s -1 ). Figure 4. Biotin-spm does not cause cytotoxicity to 293T cell. a) The morphology of viable 293T cells, examined by light microscopy at 200× magnification on a Nikon Eclipse TE2000-U stereo microscope with a digital camera attachment. The cells appeared to be unaffected by treatment with 1 mM biotin-spm for 24 h. b) The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay indicated that biotin-spm (up to 10 mM) does not alter cell viability. The cells without biotin-spm treatment was normalized as 100% .

Reagents and Chemicals
Rabbit anti-GAPDH and goat HRP-conjugated anti-mouse antibodies were purchased from Abcam and Chemicon, respectively. Mouse alkaline phosphate (AP)-conjugated anti-biotin and mouse anti-FLAG antibodies were purchased from Sigma. Immunostaining reagents such as CDP-Star Chemiluminescence reagents for AP or Western ECL reagents for HRP were purchased from Perkin-Elmer. Other chemicals were purchased from Sigma and Merck unless specified otherwise.

Synthesis of Biotinyl-spermine (biotin-spm)
The synthetic scheme is provided in supporting scheme 1. Spermine (2.0 mmol) was dissolved in excess MeOH under N 2 and the solution was then cooled to -78 o C, after which ethyl trifluoroacetate (2.0 mmol) was added dropwise over 0.5 h. The solution stirred for an additional 0.5 h before the temperature was raised to 0 o C over the next 1 h. Di-tert-butyl dicarbonate (8.0 mmol) in MeOH (20 ml) was added to the solution over 10 min at 0 o C, and stirred overnight at room temperature to protect the amino groups of spermine. The resulting mixture, without separation, was pH-adjusted to above 11 by the addition of concentrated NH 4 OH (aq) and then stirred overnight at room temperature. The resulting mixture was evaporated and purified by silica gel chromatography with MeOH/CHCl 3 /NH 4 OH (from 10/70/1 to 10/50/1), leading to a pale-yellow oily product (49% yield). The NMR 1 H and HRMS spectra were consistent with those previously reported [12] .
Biotin N-hydroxysuccinimide ester (1.58 mmol) in dry DMF is added to a suspension of N 1 ,N 2 ,N 3 -tri-Boc-spermine (1.32 mmol) in dry DMF, followed by the addition of diisopropylethylamine (1.58 mmol). The mixture was stirred overnight at room temperature and evaporated under high vacuum, and redissolved in ethyl acetate; the organic phase was sequentially washed with saturated NaHCO 3 (aq), 0.5N HCl(aq), dried over MgSO 4 , and concentrated under reduced pressure to give a glassy solid.
8 μg of pCMV2B-GspS was added to serum-free DMEM to make up a final volume of 500 μl, and 20 μl of Lipofectamine 2000 was pipetted into a separate tube of serum-free DMEM for a final volume of 500 μl. Both the plasmid and Lipofectamine were carefully and individually mixed, allowed to set for 5 min, and then mixed together. The mixtures was incubated for 20 min at room temperature to form plasmid containing liposome and then 1 ml of the mixture was slowly added dropwise into the cell culture to prevent cell detachment. The culture was carefully agitated to ensure proper dispersion and then incubated for 4 h at 37 °C with 5% CO 2 before the medium was replaced by serum-containing DMEM.

In vivo labeling and immunoblotting of Gsp-biotin in 293T cell
293T cells were subcultured into 10 cm tissue culture dishes (Corning) and transfected with the pCMV2B-GspS plasmid as previously described. After 4 h of liposome treatment, the culture media were replenished with FBS-supplemented DMEM spiked with 1 mM biotin-spm for 24 h at 37 °C, 5% CO 2 and treated with 1 mM diamide or H 2 O 2 for 10 min to enhance the level of protein glutathionylation, followed by detachment by PBS and centrifugation at 500 × g (Kobuta) for 5 min.
Cells were then sonicated in PBS. After centrifugation to remove cell debris, the resulting samples were resolved in a non-reducing SDS-PAGE. Gluthionylated proteins were detected by Western blotting using mouse alkaline-phosphatase-conjugated anti-biotin antibody. To remove biotin-spm from Gsp-biotin thiolated proteins, 10 7 cells were harvested and lysed by sonication, One half of the resulting lysates were treated with 1 μg Gsp amidase at 37 °C for 1 h, while the other half of cell lysate was treated with the same volume of SDS loading buffer containing 50 mM 2-mercaptoethanol (2-ME) and boiled for 30 min. Both the reaction mixtures were subjected to 10% non-reducing SDS-PAGE and Western blotting using an anti-biotin antibody.

Preparation and purification of GSH-modified peptides
After 4 h of liposomal transfection of pCMV2B-GspS as previously described, the were analyzed by mass spectrometry.

Mass spectrometry and data processing
The peptide mixtures from each preparation were desalted with C 18 Ziptips (Millipore), and dried in a SpeedVac (Thermo), before they were reconstituted in 5% acetonitrile and 0.1% formic acid to give a volume of 5 μl, and loaded onto a C 18 column of 75-μm × 250-mm (nanoACQUITY UPLC BEH130, Waters). The peptides mixtures were separated by online nanoflow liquid chromatography using nanoAcquity system (Waters) with a linear gradient of 5 to 50% acetonitrile (in 0.1% formic acid) in 95 min, followed by a sharp increase to 85% acetonitrile in 1 min and held for another 15 min at a constant flow rate of 300 nl min -1 . Peptides were detected in an LTQ-Orbitrap Velos hybrid mass spectrometer (Thermo Scientific) using a data-dependent HCD Top10 method. For each cycle, full-scan MS spectra ( to two missed cleavages were allowed; and mass accuracy of 10 ppm for the parent ion and 0.10 Da for the fragment ions. False discovery rates were controlled using the target-decoy strategy to distinguish correct and incorrect identifications. For the identification, the false discovery rate was set to 0.01 for peptides, proteins and sites. All mass spectrometric data of all glutathionylated proteins and peptides are available online (Supporting Information Data 1).

HPLC analysis of the intracellular Gsp
Transfected (pCMV2B-GspS) and control samples (pCMV2B vector only) were collected from culture dishes in PBS (Gibco) and centrifuged at 1,000 rpm for 5 min.
After the supernatant was decanted, the pellet was suspended in a solution of 40mM

Cell Proliferation Assay
CellTiter 96 AQ One Solution Cell Proliferation Assay (Promega) was used to determine the cytotoxicity of biotin-spm in 293T cells. pCMV2B and pCMV2B-GspS transfected cells were subcultured into 96-welled tissue-culture microtiter plates (Corning) and treated with biotin-spm at concentrations from 0 to 10 mM for 36 h, followed by addition of MTT-containing reagent at a ratio of 1:5 v/v for 1 h at 37 °C, 5% CO 2 , before measurement of the formazan absorbance. Cell images were digitally captured with 200-fold magnification by a Nikon Eclipse TE2000-U stereo microscope with a digital camera attached (Nikon).

GspS activity assay.
To 200 mM HEPES (pH 7.3) containing 1.5 mM NADH, 2 mM phosphoenolpyruvate (PEP) and 2 mM MgCl 2 was added a substrate solution of 40 mM GSH, 8 mM ATP and 0-6.4 mM spermidine (or biotin-spm), 11.4 U ml -1 pyruvate kinase, 48.96 U mL -1 lactate dehydrogenase. GspS (2.5 mg ml -1 ) was then added to initiate the enzyme activity assay. The consumption of NADH was measured by fluorescence excitation at 340 nm and emission at 460 nm to determine the degree of product formation.