Iodoacetamide ia

The equal protein from cell lysates was separated on a 12.5% SDS-polyacrylamide gel. Then, the protein in gel was cut into small pieces (~0.5–1 mm³). A small gel particle size facilitates the removal of SDS and coomassie by 25 mM NH₂HCO₃. Next, protein in gels was reduced and alkylated by dithiothreitol (DTT) for 30 min at 37 °C and iodoacetamide (IA) (Sigma-Aldrich, Darmstadt, Germany) for 30 min at room temperature in the dark, respectively. These samples were further quenched with DTT for 15 min at room temperature before incubating with trypsin at a ratio of 1:50 at 37 °C for 16 h. After digestion, tryptic proteins were extracted from the gels by adding 30 µL of 50% CH3CN/1% trifluoroacetic acid (TFA). Next, the peptides from the supernatant were removed and collected in a clean LoBind tube and the extracts were concentrated on speedvac. Finally, these digested proteins were resuspended in 0.1% formic acid and subjected to LC-MS/MS (Thermo Scientific, Waltham, MA, USA). The data from mass spectrometry were analyzed by Proteome Discoverer version 2.1softwere (Thermo Scientific, Waltham, MA, USA).

A previously established rodent model of FD was used in this experiment, described as follows [24 (link)]: After a three-day period of acclimation, ten-day-old pups were randomly divided into two groups: one group (FD) was given a gavage of 0.2 ml of 0.1% iodoacetamide (IA, Sigma, US) in 2% sucrose (Sigma,US), while the other group (control) was given only 0.2 ml of 2% sucrose. All pups were sent to their mother rats immediately after gavage and fed normally until they were seven weeks old. During the course, the animals were monitored daily for their overall behaviors, including food intake, activity level, alertness and feces.