Mouse lines and tissues

All animal-related experiments were conducted in accordance with the Canadian Council on Animal Care Standards and the Animals for Research Act and were approved by the University of Ottawa Animal Care Council. WT C57BL/6 J mice were purchased from the Jackson Laboratory. The prkn−/− mice (C57BL/6 J background) were obtained from Dr. A. Brice [19]. The Sod2± mice (C57BL/6 J) were generated by Lebovitz [26], and purchased from the Jackson Laboratory; prkn−/−//Sod2+/+ and Sod2±//prkn+/+ mice were crossed to generate prkn±//Sod2± offspring, which was interbred to produce the desired prkn−/−//Sod2± (bi-genic) mouse. Brains and hearts were collected for the four genotypes of interest: WT; prkn−/−; Sod2±; and prkn−/− //Sod2±.

Genotyping

Ear tissue was collected for genotyping. DNA was extracted from the tissue by incubating the ear sample in 1× solution A (Solution A (10×): 250 mM NaOH, 2 mM EDTA, in water) at 95 °C for 30 min, followed by neutralizing the reaction with 1× solution B (Solution B (10×): 400 mM Tris-HCl, in water). Standard polymerase chain reaction (PCR) was used to amplify the prkn and Sod2 loci. The following primers were used:

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Amplification products were electrophoresed on a 1% agarose gel and stained with ethidium bromide. A band at 300 base pairs (bp) represented a prkn+/+ or prkn−/− amplification product (where a sample with 300 bp bands for both prkn+/+ and prkn−/− primer pairs was identified as a prkn± mouse). A band at 123 bp represented a Sod2+ genotype and a band at 240 bp represented a Sod2− allele.

SOD activity assay

A superoxide dismutase (SOD) assay kit (Cayman chemical) was used to measure MnSOD (SOD2) activity in mouse brain. Pre-weighed, perfused mouse brain pieces were homogenized in 5 mL cold 20 mM HEPES buffer, pH 7.2, supplemented with EGTA, mannitol and sucrose, with a Dounce homogenizer on ice. The homogenates were centrifuged at 1500×g 5 min at 4 °C. The resulting supernatants were centrifuged at 10,000×g 15 min at 4 °C to isolate the mitochondria (pellet). Two mM potassium cyanide, which inhibits Cu/Zn-SOD and extracellular SOD, was added to each sample to ensure assay specificity for MnSOD. The SOD standards were prepared by adding 200 μL of the radical detector and 10 μL of the provided standards, in duplicates in a 96-well plate. The same was repeated for the samples. The reaction was initiated by adding 20 μL of xanthine oxidase to all the wells. Background absorbance was assayed by adding 20 μL xanthine oxidase to sample buffer (optional). The plate was incubated on a shaker for 30 min at room temperature. The absorbance was measured at 450 nm. The linearized SOD standard curve was plotted and used to calculate MnSOD activity (U/mL) from averaged sample absorbance readings.

Measurements of reactive oxygen species (ROS) in tissues

The Amplex® Red hydrogen peroxide/peroxidase assay kit (Invitrogen) was used to monitor endogenous levels of H2O2 in mouse tissues and cells. Pre-weighed hearts and specimens of cortices as well as midbrains (or pelleted cells) were homogenized on ice in the 1× reaction buffer provided, using a Dounce homogenizer (3 times volume to weight ratio). Homogenates were diluted in the same 1× reaction buffer (10× and 5×). A serial dilution of the H2O2 standard provided was prepared (20, 10, 2 and 0 μM). Fifty μL of standards and samples were plated in a 96 well black plate with clear flat bottom. The reaction was started by the addition of 50 μL working solution which consisted of 1× reaction buffer, Amplex® red and horseradish peroxidase. The plate was incubated at room temperature for 30 min protected from light. A microplate reader was used to measure either fluorescence with excitation at 560 nm and emission at 590 nm, or absorbance at 560 nm. The obtained H2O2 levels (μM) were normalized to tissue weight (in g) or protein concentration (μg/μL).

ROS measurements in intact cells

Mammalian cells, including SH-SY5Y and HEK293 cells, were transfected with flag-tagged WT PRKN cDNA or control vector (pcDNA), as described before. After 24 h the cells were lifted using trypsin and re-seeded in a 12-well dish at a density of 0.3 × 106 cells/mL. After 48 h the cells were treated with 0 mM or 2 mM H2O2 in OPTI-MEM medium at 37 °C and 5% CO2. After 1 h the cells were washed with OPTI-MEM and incubated with 20 μM of dichlorofluorescin diacetate (DCFH-DA, Sigma) for 30 min at 37 °C and 5% CO2. Cells were collected using a cell lifter and treated with ethidium-1 dead stain (Invitrogen) for 15 min at room temperature. Samples were analyzed using a BD Fortessa flow cytometer set to measure ROS-sensitive signals (DCFH-DA, ex. 488 nm and em. 527 nm) and viability-related stains (ethidium-1, ex, 528 nm and em. 617 nm). The results were reported as the average mean fluorescence intensity (MFI) of ROS in live cells. Each separate transfection was considered one biological replicate.

Western blotting and densitometry

Brain and heart homogenates as well as cell lysates were run on 4–12% Bis–Tris SDS-PAGE gels using MES running buffer. Proteins were transferred to PVDF membranes using transfer buffer, and immunoblotted for parkin, DJ-1, MnSOD, Aco2, mitochondrial creatine kinase (mtCK), VDAC, TOM20, nitrotyrosine, glutathione reductase, and glyoxalase-1. Actin and Ponceau S staining were used as loading controls. For densitometry quantification, the signal intensity of protein nitrotyrosination and glutathione reductase from each sample was measured as pixel using Image J Software and controlled for total protein loading.

Protein carbonyl assay

A protein carbonyl colorimetric assay kit (Cayman chemical) was used to assay the carbonyl content in human and mouse brains or hearts. Pre-weighed tissues were rinsed in PBS and then homogenized in 1 mL cold PBS at pH 6.7 supplemented with 1 mM EDTA, using a Dounce homogenizer on ice. Homogenates were centrifuged at 10,000×g for 15 min at 4 °C. Two hundred μL of the supernatant was added to a tube with 800 μL 2,4-Dinitrophenylhydrazine (DNPH; sample tube) and 200 μL of the supernatant was added to a tube with 800 μL 2.5 M HCl (control tube), both tubes were incubated in the dark for 1 h with occasional vortexing. 1 mL 20% TCA followed by 1 mL 10% TCA solutions were added to the centrifuged (10,000×g 10 min at 4 °C) pellet after discarding the supernatant. The resulting pellet was resuspended in 1 mL of 1:1 ethanol:ethyl acetate mixture and centrifuged 3 times to extract protein pellets. The final pellets were suspended in 500 μL guanidine hydrochloride and centrifuged. A total of 220 μL per sample and control supernatants were added to two wells of a 96-well plate, and the absorbance was measure at 360 nm. The corrected absorbance (CA, sample value minus control value) was used in the following equation to obtain the protein carbonyl concentration: Protein Carbonyl (nmol/mL) = [(CA)/(0.011 μM−1)](500 μL/200 μL). Total protein concentration from the sample tissues was measured to obtain the carbonyl content, i.e., protein carbonyl/total protein concentration.

Cell cultures, transfection and oxidation

Mammalian cell cultures (CHO; HEK293; SH-SY5Y) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C with 5% CO2. For transient transfection paradigms, 4 to 15 μg of cDNA coding for N-terminally Flag-tagged WT human parkin or empty Flag control vector (pcDNA3.1) using a 1:1 ratio of cDNA:Lipofectamine 2000, was used for ectopic expression. The cDNA and Lipofectamine 2000 reagent were incubated for 20 min at RT before being applied to the cells for 1 h at 37 °C with 5% CO2, followed by direct addition of fresh culture medium. Cells were incubated another 24 h before treatment, harvesting and analysis. Chinese hamster ovary cells, stably expressing the myc-vector (CHO), or N-terminal myc-tagged, WT human parkin (CHO-parkin) were also used [26].

All chemicals (H2O2, DTT, BSO and NAC) were added directly to intact cells at ~ 75% confluence in growth or OPTI-MEM media. Cells were manually scraped, spun at a 100×g for 5 min, the pellets washed with PBS and then homogenized in a Tris salt buffer, transferred to ultracentrifuge tubes and spun at 163, 200×g and 4 °C for 30 min to extract the soluble fraction. The resulting pellets were further homogenized in the Tris salt buffer with the addition of 2–10% SDS, transferred to ultracentrifuge tubes and spun at 163, 200×g and 10 °C for 30 min to extract the insoluble fraction. SH-SY5Y cells were seeded at a density of 0.5–1 × 106 cells/mL. Once cells reached 70–80% confluency they were transfected with cDNA coding for C-terminally flag-tagged parkin or empty flag control vector (pcDNA3) by electroporation using the nucleofector method described by Hu and Li, 2015. A total of 2 million cells were resuspended in 100 μL of OPTI-MEM containing cDNA (2 μg) and 1% polyoxamer 188. The cells were electroporated using the X Unit and pulse code “CA-137” on a Lonza 4D-Nucleofector. Following electroporation, cells were seeded at a concentration of 0.8–1 × 106 cells/mL [18].

Expression of recombinant, maltose-binding protein-tagged parkin

WT and truncated (amino acid residues 327–465) human parkin proteins were generated in the pMAL-2 T vector (a gift from Dr. Keiji Tanaka), as previously described [29]. Parkin produced in this vector contained an N-terminal maltose-binding protein (MBP) and a thrombin cleavage site (LVPRGS). All proteins were overexpressed in E. coli BL21 Codon-Plus competent cells (New England Biolabs) and grown at 37 °C in 2% Luria Broth containing 0.2% glucose and 100 mg/L ampicillin until OD600 reached 0.3–0.37, at which point protein expression was induced with addition of 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cultures were left to express protein at 37 °C until OD600 reached 0.9–1.0. Harvested protein isolates were purified using amylose resin in buffers containing 100 μM zinc sulfate and 10 mM maltose.

Expression of recombinant, tag-less parkin

WT human parkin was generated as an initially 6His-Smt3-tagged protein in Escherichia coli BL21 (DE3) Codon-Plus RIL competent cells (New England Biolabs), as described [3, 24, 50, 53]. Transformed bacteria were grown at 37 °C in 2% Luria Broth containing 30 mg/L kanamycin until OD600 reached 0.6, at which point the temperature was reduced to 16 °C. Parkin protein-expressing cultures were supplemented with 0.5 mM ZnCl2. Once OD600 reached 0.8, protein expression was induced with IPTG, except for ULP1 protease expression, which was induced once OD600 had reached 1.2. The concentration of IPTG used for each construct is as follows: 25 μM for WT parkin, and 0.75 mM for the ULP1 protease, as described in detail. Cultures were left to express protein for 16–20 h. Cells were harvested by centrifugation, lysed and processed via Ni–NTA agarose beads in elution columns. ULP1 was purified in a similar fashion and used to cleave the tag off of the 6His-Smt3-parkin fusion protein, thus generating full-length r-parkin (aa1-465), as described [11, 53].

Cell cytotoxicity assay

A Vybrant™ cytotoxicity assay kit (Molecular Probes V-23111) was used to monitor cell death through the release of the cytosolic enzyme glucose 6-phosphate dehydrogenase (G6PD) from damaged cells into the surrounding medium. 50 μL of media alone (no cells), media from control and stressed CHO-parkin and control cells and cell lysates were added to a 96-well microplate. 50 μL of reaction mixture, containing reaction buffer, reaction mixture and resazurin, was added to all wells, and the microplate was incubated at 37 °C for 30 min. A microplate reader was used to measure resorufin fluorescence with excitation at 560 nm and emission at 590 nm. A rise in fluorescence indicates a rise in G6PD levels, i.e., a rise in cell death.

Isolation of mitochondria from tissues

Freshly dissected brain tissue was cut into smaller pieces, rinsed in cold PBS, and homogenized using either a Dounce homogenizer or Waring blender in the presence of twice the tissue volume of buffer A (20 mM Hepes pH 7.4, 220 mM mannitol, 68 mM sucrose, 80 mM KCl, 0.5 mM EGTA, 2 mM Mg(Ac)2, 1 mM DTT, 1× protease inhibitor (Roche)). The sample was centrifuged at 4070×g in a tabletop centrifuge for 20 min at 4 °C. The supernatant was collected and spun again as above. The resulting supernatant was spun at 10,000×g for 20 min at 4 °C and the pellet was then washed in the above buffer and spun again at 10,000×g for 20 min. The resulting mitochondrial pellet was resuspended in buffer B (20 mM Hepes pH 7.4, 220 mM mannitol, 68 mM sucrose, 80 mM KCl, 0.5 mM EGTA, 2 mM Mg(Ac)2, 10% glycerol), aliquoted, and snap frozen.

Aconitase assay

The Aconitase Enzyme Activity Microplate Assay Kit (MitoSciences) was used to measure activity in mitochondria isolated from mouse brain as per manufacturer’s instructions. Two brains each from 12 month-old mice were pooled to provide enough mitochondria and normalized for total protein concentration. These were treated with 0 or 4 µM H2O2 just prior to assay. The catalytic conversion of isocitrate to cis-aconitate by aconitase was measured by quantifying the amount of cis-aconitate in the reaction by reading the samples at 240 nm. Rates in µM/min were determined from 3 independent experiments performed in triplicate.

Creatine kinase assay

The EnzyChrom Creatine Kinase Assay Kit (BioAssay Systems) was applied to measure activity in mouse brain mitochondria (as above). Two brains each from 12 month-old mice were pooled to provide enough mitochondria and normalized for total protein concentration. Mitochondria were incubated with 0 or 0.5 mM H2O2 at room temperature for 20 min prior to assay start. The creatine kinase-dependent catalytic conversion of creatine phosphate and ADP to creatine and ATP was quantified indirectly by measuring NADPH at 340 nm. ATP produced by the reaction phosphorylates glucose to glucose-6-phosphate (G6P) by hexokinase, which is oxidized by NADP+ in the presence of G6P-dehydrogenase, yielding NADPH. Rates in uM/min were calculated for 3 independent experiments done in triplicate.

Glutathione quantification by HPLC

Human and mouse brain specimens as well as pelleted CHO cells were homogenized in buffer containing 125 mM sucrose, 5 mM TRIS, 1.5 mM EDTA, 0.5% trifluoroacetic acid (TFA) and 0.5% mycophenolic acid (MPA) in mobile phase. Samples were spun at 14,000×g at 4 °C for 20 min. Supernatants were collected and analyzed using an Agilent HPLC system equipped with a Pursuit C18 column (150 × 4.6 mm, 5 µm; Agilent Technologies) operating at a flow rate of 1 mL/min. The mobile phase consisted of 0.09% TFA diluted in ddH2O and mixed with HPLC-grade methanol in a 90:10 ratio. Standard solutions were used to estimate the retention times for GSH and GSSG. Using Agilent Chemstation software, the absolute amounts of GSH and GSSG were calculated by integrating the area under the corresponding peaks, and values were calculated from standard curves.

Glutathione concentration determined by monochlorobimane assay

Stock solutions of assay dye (monochlorobimane (MCB), 22 mM) and glutathione-S-transferase (50 units/mL) were prepared in PBS and stored protected from light at − 20 °C. The working solution was prepared using 12.8 μL of stock MCB and 80 μL of stock glutathione-S-transferase in 4 mL PBS and stored on ice. Samples were prepared as follows: cells were lifted mechanically using cell-lifters, washed twice and re-suspended in ice-cold PBS, mixed by vortex and incubated on ice for 30 min. Following two freeze thaw cycles using solid CO2, the samples were sonicated 1 min on wet ice (S220 Ultra-sonicator from Covaris) and spun at 3000×g, 4 °C, for 5 min. Total protein concentration of supernatants was determined using Bradford assay. Samples and glutathione (GSH) standards (0–13 μM) were plated in 25 μL aliquots in a 96-well plate with clear bottom and black sides. Twenty-five μL of working solution was added to all experimental wells and protected from light for 15 min at room temperature. Fluorescence (ex 380 nm, em 461 nm) was measured using a Synergy H1Multi-Mode Plate Reader (Bio Tek). The amount of GSH detected in each sample was calculated using the regression curve obtained from the glutathione standards.

Tietze’s enzymatic recycling method to quantify glutathione

The enzymatic recycling method described by Rahman et al. [39] was used to determine GSH and GSSG levels in mouse brain lysates. Hemibrains of WT (n = 3) and prkn knock-out (n = 3) mice, at ~ 12 months of age respectively, were collected, weighed and homogenized in 3× v/w of KPE-X (0.1 M potassium phosphate, 5 mM EDTA, 0.1% Triton X-100, 0.6% sulfosalicylic acid, pH 7.5) using a glass Dounce homogenizer (50 passes). Samples were spun at 8000×g at 4 °C, for 5 min and the supernatant protein concentration was determined using a Bradford assay. To determine the total glutathione (GSH + 2GSSG) concentration, the following stock solutions were freshly prepared in KPE (0.1 M potassium phosphate, 5 mM EDTA, pH 7.5): 5,5ʹ-dithio-bis-2-nitrobenzoic acid (DNTB) at 0.6 mg/mL, NADPH at 0.6 mg/mL and glutathione reductase at 3 units/mL. GSH standards were prepared in KPE at concentrations of 0–26 nM/mL. Twenty μL of diluted sample or GSH standard was added per well and 120 μL of a 1:1 mixture of the DNTB and glutathione reductase stocks solutions was added to each assayed well. After 30 s incubation, 60 μL of the NADPH was added and absorbance was immediately measured at 412 nm in 30 s intervals for a total of 2 min.

To determine the level of GSSG, samples were diluted (1:4) in KPE and treated with 0.2% 2-vinylpyridine for 1 h at RT. Excess vinyl-pyridine was quenched with 1% triethanolamine and GSSG was measured using the same method as total glutathione except for: GSH standards were replaced with GSSG (0 to 26.24 nM/mL) treated with vinyl-pyridine and triethanolamine. Absolute values for total glutathione (GSH + 2GSSG) and GSSG per sample were calculated using the linear regression obtained from the change in absorbance/min plotted against GSH or GSSG standards, respectively, divided by the total protein concentration. Absolute values for GSH were determined using the equation: GSH = [GSH + GSSG] − 2 [GSSG] [39].

Total RNA isolation, cDNA synthesis and PCR-based amplification

Pre-weighed, murine cortices were homogenized in QIAzol (Qiagen), at 1 mL volume per 100 mg of tissue, and incubated at room temperature for 5 min. 0.2 mL of chloroform per 1 mL QIAzol was added and the homogenates were shaken vigorously for 15 s, followed by a 2–3 min incubation at RT, the tubes were centrifuged at 12,000×g for 15 min at 4 °C. The upper clear aqueous layer was transferred to a new tube and 1 volume of 70% ethanol was added and mixed by vortexing. The solution was then added to an RNAeasy Mini spin column (Qiagen) placed in a 2 mL collection tube and centrifuged for 15 s at 8000×g at RT. The flow-through was discarded and 700 μL Buffer RW1 was added to the spin column and spun for 15 s at 8000×g. The same step was repeated with 500 μL Buffer RPE, one spin for 15 s and a second spin for 2 min. An optional spin in a new collection tube at full speed for 1 min to remove excess buffer was added. The RNAeasy Mini spin column was placed in a new collection tube, 50 μL RNase-free water was added directly to the membrane and centrifuged for 1 min at 8000×g. A NanoDrop machine was used to measure the amount of total RNA obtained from the cortices. Turbo DNA-free™ (Life Technologies) was used to remove trace to moderate amounts of contaminating DNA. SuperScripT™ IV First-Strand Synthesis System (Invitrogen) was used for cDNA synthesis reaction. iTaq™ Universal SYBR® Green Supermix (BIO-RAD) and select primer sets were used for PCR amplification of the newly synthesized cDNA templates, and controls, and analyzed by agarose gel electrophoresis and ethidium bromide staining. The following primers were used [9, 44]:

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Glutathione reductase activity assay

A glutathione reductase (GR) enzyme assay kit (Cayman Chemical) was used to measure its activity in tissues. Human and mouse brains were either perfused or rinsed with PBS (pH 7.4). Tissues were homogenized in 5–10 mL of cold buffer (50 mM potassium phosphate, pH 7.5, 1 mM EDTA) per gram of tissue, followed by centrifugation at 10,000×g for 15 min at 4 °C. In a 96 well clear plates, three wells were loaded with 120 μL assay buffer (provided) and 20 μL GSSG (provided) as background control; and three wells were loaded with 100 μL assay buffer, 20 μL GSSG and 20 μL diluted GR (provided) as positive control. Twenty μL of samples supernatant were loaded in triplicates, with 100 μL assay buffer and 20 μL GSSG. Reactions were initiated by adding 50 μL NADPH (provided) to each well. The 96-well plate was gently shaken for a few seconds and absorbance was read at 340 nm once every minute for 10 min, or to obtain readings at a minimum of 5 time points. The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm and is directly proportional to the GR activity in the sample.

Parkin-mediated redox recycling of glutathione

Parkin protein buffer exchange to T200 protein buffer (50 mM Tris, 200 mM NaCl, pH 7.5) was first performed using repeat centrifugations (8 times 4000×g at 4 °C for 10 min) in Amicon Ultra 10 kDa MWCO filters. Protein concentration was adjusted to 10 μM using T200. Both GSH and GSSG stocks were prepared in PBS at concentrations of 1 mg/mL (3250 μM) and 2.01 mg/mL (6560 μM) respectively. Glutathione standards of 0, 2.5, 5, 10 μM and 100 μM of both GSH and GSSG were prepared and combined in the following ratios to a final volume of 90 μL: 10 μM GSH: 0 μM GSSG, 9 μM GSH: 1 μM GSSG, 8 μM GSH: 2 μM GSSG, 6 μM GSH: 4 μM GSSG, 4 μM GSH: 6 μM GSSG, 2 μM GSH: 8 μM GSSG, 1 μM GSH: 9 μM GSSG, and 0 μM GSH: 10 μM GSSG. Full-length r-parkin (1 μL of a 10 μM solution) was added to the prepared mixtures and allowed to incubate at room temperature for 15 min. Samples were analyzed for GSH concentration using the monochlorobimane assay described above.

Glutathionylation assays

S-glutathionylation of recombinant parkin proteins was performed, as described previously [6]. MBP-tagged parkin proteins were eluted from columns with excess maltose. Concentrated eluates were supplemented with 0.1% DMSO (10 µL DMSO in 10 mL PBS), and excess DTT and maltose were removed by several cycles of centrifugation with 30 kDa cut-off filters. Proteins/peptides (at 14 µM) were incubated with 3 mM GSH for 1 h and then with 5 mM GSSG for 2 h at room temperature. Trypsin digestion was performed (Peptide:Trypsin = 20:1) overnight at 4 °C. Trypsin-digested fragments were run through MALDI analysis. To monitor S-glutathionylation, eosin-labeled GSSG (Di-E-GSSG) was used to glutathionylate proteins, as described [43]. Di-E-GSSG has quenched fluorescence in the disulphide form. Fluorescence increases ~ 20-fold upon reduction of its disulphide bond following the formation of E-GSH. Blackened 96-well-plates were used in a PerkinElmer Victor3 multilabel counter containing a final well volume of 200 μL in 0.1 M potassium phosphate buffer (pH 7.5), 1 mM EDTA. The reaction was started by addition of 20 μM Di-E-GSSG to parkin proteins, followed by recording the fluorescence emission at 545 nm after excitation at 520 nm. Controls with no peptide added were used as fluorescent background.

To confirm S-glutathionylation, reaction products were tested for possible deglutathionylation. Aliquots of S-glutathionylated proteins were treated with 10 mM DTT or with the complete GSH-glutaredoxin (Grx) system. All samples were run on a non-reducing SDS-PAGE, containing 4–12% acrylamide. The gel was exposed to UV transilluminator to visualize eosin-tagged glutathionylated protein. The same gels were later stained with Coomassie Blue dye. Di-Eosin-GSSG was purchased from IMCO or Cayman Chemical (11,547), Sweden. Human Grx-1, and Grx-2 were prepared, as described [43]. Rat recombinant thioredoxin was a kind gift from Prof. Elias Arner.

Detection of S-glutathionylated parkin in cultured cells

S-glutathionylation of parkin in cells was performed using a modified BioGEE protocol described by Sullivan et al. [51]. CHO cells stably expressing myc-parkin [25] were treated with or without 20 µM BioGEE (Invitrogen G36000) for 3 h with the addition of 1 mM H2O2 to the media for the final 10 min. Cells were collected, pelleted and washed once with ice-cold 1× PBS, followed by two washes with 1× PBS + 50 mM iodoacetamine (IAA) (BioRad) with 5 min of rocking at RT for the second wash. The cells were then lysed in 200 µL 1× RIPA buffer + 50 mM IAA and incubated on ice for 30 min followed by a snap freeze. Lysates were thawed on ice, sonicated twice for 20 s, centrifuged at 14,000×g for 10 min at 4 °C, and protein concentration was equalized. The lysates were pre-cleared by incubation with biotin-blocked streptavidin-conjugated magnetic beads. For this, 50 µL of beads (Pierce) were blocked with free D-Biotin (Novabiochem) (3 mg/mL prepared in alkaline water (pH 10.5)) and rocked for 1 h at RT, followed by 5 washes with 1× PBS and incubation with the cell lysates (50 µL) for 30 min at 4 °C with rocking to remove non-specific binding of proteins to the bead complex. The pre-cleared lysate was then incubated with 50 µL of pre-washed (unblocked) streptavidin-conjugated beads and rocked for 1 h at 4 °C. The beads were washed 5 times with 10× volume of ice-cold RIPA buffer, twice with 10× volume of 0.1% SDS in PBS and then resuspended in 1 volume of 0.1% SDS/PBS and incubated for 30 min at RT. Supernatant from this incubation was saved and labelled “-DTT eluate”. Beads were then resuspended in 1 volume of 0.1% SDS in PBS containing 10 mM DTT, and rocked for 30 min at RT. The supernatant was saved and labelled “ + DTT eluate”. The eluates were passed through a 10 kDA cut-off filter and concentrated by centrifugation at 14,000×g for 20 min at 4 °C; the retentate was collected by centrifugation at 1000×g for 5 min at 4 °C. Samples were then resolved by SDS-PAGE using a 10% gel under reducing conditions. Parkin was detected by Western blot analysis using a polyclonal anti-parkin antibody (Cell Signaling, 2132S).

Mass spectrometry analysis of S-glutathionylated parkin

S-glutathionylated proteins were treated with trypsin and the resulting peptides were separated using one dimension of liquid chromatography (LC). The LC eluent was interfaced to a mass spectrometer using electrospray ionization and peptides were analyzed by MS. LC–MS/MS analyses were performed using an Easy-nLC chromatography system directly coupled online to a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer with a Thermo Scientific™ Nanospray Flex™ ion source. The sample was injected from a cooled autosampler onto a 10 cm long fused silica tip column (SilicaTips, New Objective, USA) packed in-house with 1.9 μm C18-AQ ReproSil-Pur (Dr. Maisch, Germany). The chromatographic separation was achieved using an acetonitrile (ACN)/water solvent system containing 0.1% formic acid and a gradient of 60 min from 5 to 35% of ACN. The flow rate during the gradient was 300 nL/min. MS/MS data were extracted and searched against in-house Mascot Server (Revision 2.5.0), a search engine that uses mass spectrometry data to identify and characterize proteins from sequence databases. The following parameters were used: trypsin digestion with a maximum of two missed cleavages; Carbamidomethyl (–C), Oxidation (–M), Deamidated (–NQ) and Glutathione (–SG) as variable modifications; and a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da. The identified protein was filtered using 1% false discovery rate and at least two peptides per protein as limiting parameters.

Statistical analyses

All statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software www.graphpad.com). Differences between two groups were assessed using a Student’s t-test. Differences among 3 or more groups were assessed using a 1-way or 2-way ANOVA followed by Tukey or Dunnett post hoc corrections (as indicated) to identify statistical significance. Subsequent post hoc tests are depicted graphically and show significance between treatments. For all statistical analyses, a cut-off for significance was set at 0.05. Data are displayed with p values represented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.