Monospin c18 column

Extracted proteins (100 µg) in lysis buffer were adjusted to a final volume of 100 µL. Methanol (400 µL) was added to each sample and mixed before the addition of 100 µL of chloroform and 300 µL of water. After mixing and centrifugation at 20,000× g for 10 min to achieve phase separation, the upper phase was discarded and 300 µL of methanol was added to the lower phase, and then centrifuged at 20,000× g for 10 min. The pellet was collected as the soluble fraction [68 (link)].
Proteins were resuspended in 50 mM NH₄HCO₃, reduced with 50 mM dithiothreitol for 30 min at 56 °C, and alkylated with 50 mM iodoacetamide for 30 min at 37 °C in the dark. Alkylated proteins were digested with trypsin and lysyl endopeptidase (Wako, Osaka, Japan) at a 1:100 enzyme/protein ratio for 16 h at 37 °C. Peptides were desalted with Mono Spin C18 Column (GL Sciences, Tokyo, Japan). Peptides were acidified with formic acid (pH < 3) and analyzed by nano-liquid chromatography (LC) mass spectrometry (MS)/MS.

Blood and urine were collected from isoflurane-anaesthetized mice through cardiac and urinary bladder punctures, respectively. Serum (or urinary) levels of total protein, creatinine, urea nitrogen, AST, and ALT were measured using commercial colorimetric assay kits from Fujifilm-Wako (Osaka, Japan). For the blood oxytocin measurements, blood was collected from the retro-orbital plexus. Serum was prepared and its protein components were denatured by 0.05% trifluoroacetic acid and removed by the MonoSpin C18 column (GL Science, Tokyo, Japan) with acetonitrile elution. After acetonitrile evaporation, oxytocin contents were measured using the Oxytocin ELISA Kit (Enzo Life Sciences, CA, NY, USA) according to the manufacturer’s instruction.

In vitro kinase assays of GST‐tagged AKT1 and Flag‐tagged SOD2 WT or S27A were done, unlabeled ATP was used in the reactions. Then the reactions were terminated by freezing and processed for mass spectrometry. In brief, proteins were precipitated with acetone and the protein pellets were dried by using a SpeedVac for 1–2 min. The pellets were subsequently dissolved in 8 m urea, 100 mm tris‐HCl, pH 8.5. 5 mm TCEP (Thermo Scientific), and 10 mm iodoacetamide (Sigma) and alkylation were added to the solution and incubated at room temperature for 30 min, respectively. The protein mixtures were diluted four times and digested overnight with Trypsin at 1:50 w/w (Promega). The digested peptide solutions were desalted using a MonoSpin C18 column (GL Science, Tokyo, Japan) and dried with a SpeedVac.
The peptide mixtures were analyzed by LC/tandem MS (MS/MS). Data‐dependent tandem mass spectrometry (MS/MS) analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). The acquired MS/MS data were analyzed against a Swiss‐Prot Homo sapiens database using PEAKS Studio 8.5 (Bioinformatics Solutions, Waterloo, Ontario, Canada). The database search parameters were set as the followings: MS and MS/MS tolerance of 20 ppm and 0.1 Da, respectively, FDR was set as 1% and protein identification threshold was set as (−10 logP) ≧ 20.