Sep pak

Unless otherwise noted, proteomics and data analysis was performed as described (Ordureau et al., 2020 (link)). Briefly, protein extracts (100 μg) were subjected to disulfide bond reduction with 5 mM TCEP (room temperature, 10 min) and alkylation with 25 mM chloroacetamide (room temperature, 20 min). Methanol–chloroform precipitation was performed prior to protease digestion. In brief, four parts of neat methanol were added to each sample and vortexed, one part chloroform was then added to the sample and vortexed, and finally three parts water was added to the sample and vortexed. The sample was centrifuged at 6 000 rpm for 2 min at room temperature and subsequently washed twice with 100% methanol. Samples were resuspended in 100 mM EPPS pH8.5 containing 0.1% RapiGest and digested at 37°C for 8h with Trypsin at a 100:1 protein-to-protease ratio. Samples were acidified with 1% Formic Acid for 15 min and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters).

Kidney tissues were lysed with a mechanical homogenizer using lysis buffer composed of 50 mM HEPES pH 8.5, 250 mM NaCl, and EDTA-free protease inhibitor cocktail (Roche), 5 mM TCEP in 2 % SDS. Kidney lysates were centrifuged at 10,000 × g for 10 minutes. Protein content was measured using a BCA assay (Thermo Scientific); disulfide bonds were reduced and cysteine residues alkylated with iodoacetamide (14 mM) essentially as previously described (Huttlin et al., 2010 (link)). Protein lysates were purified by methanol-chloroform precipitation and 200 μg was digested overnight with LysC (Wako, Japan) in a ½00 enzyme/protein ratio in 2 M urea and 25 mM HEPES, pH 8.5. Digests were acidified with 10% acetic acid (AA) to a pH of ~ 2 and subjected to C18 solid-phase extraction (50 mg SPE) (Sep–Pak, Waters).
Isobaric labeling of the peptides was performed using 10-plex tandem mass tag (TMT) reagents (Thermo Fisher Scientific). Reagents, 5.0 mg, were dissolved in 252 μl acetonitrile (ACN) and 1/10 of the solution were added to 100 μg of peptides dissolved in 100 μl of 200 mM HEPES, pH 8.5. After 1 hour (RT), the reaction was quenched by adding 3 μl of 5% hydroxylamine. Labeled peptides were combined and acidified prior to C18 SPE on Sep-Pak cartridges (50 mg).

TMT reagents (0.8 mg) were dissolved in anhydrous acetonitrile (40 μL) of which 10 μL was added to the peptides (100 μg) with 30 μL of acetonitrile to achieve a final acetonitrile concentration of approximately 30% (v/v). Following incubation at room temperature for 1 h, the reaction was quenched with hydroxylamine to a final concentration of 0.3% (v/v). The TMT-labeled samples were pooled at a 1:1:1:1:1:1:1:1:1:1 ratio across the 10 samples. The pooled sample was vacuum centrifuged to near dryness and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters).

A solution of deferoxamine mesylate (0.099 mmol, 64.9 mg) and triethylamine (20.5 μL, 0.15 mmol) in 0.25 mL dimethyl sulfoxide was added to 3 (15.1 mg, 0.025 mmol) in 0.1 mL of dimethyl sulfoxide. After stirring for 30 min the reaction was loaded directly on a 5 g C₁₈ Waters Sep-Pak and purified (compound 4 eluted off with H₂O:MeCN 17:7) to give 4 (12.1 mg, 46.5%). ¹H NMR (400 MHz, CDCl₃) δ 8.73 (m, 2H), 7.53 (m, 2H), 5.98 (s, 2H), 3.61 (m, 5H), 3.51 (m, 1H), 3.17 (m, 4H), 3.12 (s, 3H), 2.79 (m, 2H), 2.59 (m, 4H), 2.54 (s, 6H), 2.32 (m, 2H), 2.17 (m, 2H), 2.09 (m, 2H), 1.98 (m, 2H), 1.56 (bs, 20H), 1.43 (s, 6H), 1.42 (s, 3H). ESIMS [M–H]⁻ calcd for C₅₂H₇₃BFN₁₂O₁₀ 1055.57, found 1055.80.