Mmts solution

Mitochondrial protein quantification was then performed by BCA. The purified mitochondria were washed twice in PBS, and lysed in RIPA lysis buffer (CWBIO, China) supplemented with protease inhibitors. The lysate was heated to 60 °C for 1 h in 0.05 M TCEP solution (Sigma, 646,547) and then was placed at room temperature away from light in 55 mM MMTS solution (Sigma, 208,795). The lysate was added to a 10 KDa ultrafiltration tube (Pall, OD 010C33) and centrifuged at 12000 g for 20 min. Next, 100 ul of UA (8 M urea pH 8.5, Amresco, 0568) solution was added to the lysate and then centrifuged at 12000 g for 20 min, twice. One hundred microliter 0.25 M TEAB solution was added to the lysate and then centrifuged at 12000 g for 20 min. The entire centrifugation process was repeated three times. Then, 2% trypsin (Promega, V5280) was added to the sample, and incubation was conducted at 37 degrees overnight. The next day, booster digestion was performed using an additional dose of trypsin. After digestion, the peptides were dissolved in sample solution (0.1% formic acid and 2% acetonitrile) and centrifuged at 13200 rpm for 4 min; then, mass spectrometry was used to identify the supernatant.

The protein concentrations were assessed a second time after trichloroacetic acid (TCA) precipitation overnight at 4°C using 1/3 volume of TCA, to ensure correct protein levels. For each sample 100 μg of protein was reduced using 5mM Tris-2-(carboxyethyl)-phosphine hydrochloride solution (TCEP; Pierce Biotechnology) and cysteine blocking was performed with 2mM methyl methanethiosulphonate (MMTS) solution (Sigma-Aldrich). Each sample containing 100 μg proteins, was digested using 10 μg trypsin (Promega) at 37°C overnight. Samples were then labelled using iTRAQ reagents (ABSciex) before being pooled into one mixture. The following iTRAQ labels were used for the transfected cells: iTRAQ labels 113 was used for the transfected cells with the pcDNA3.1+ empty plasmid; iTRAQ labels from 114 to 118 were used to label the differentially transfected cells with RXFP3 (114-0.5 μg_RXFP3-HA, 115-1 μg_RXFP3-HA, 116-2 μg_RXFP3-HA1, 117-5 μg_RXFP3-HA, 118-10 μg_RXFP3-HA). After labelling, the samples were pooled, dried down and dissolved with SCX buffer prior to SCX chromatography.