2.2. Sample Preparation for Proteomic Analysis

Protein sample preparation and shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed at Purdue University’s Proteomics Core. To start, frozen tissues were thawed and homogenized in 100 mM ammonium bicarbonate buffer at 6500 rpm for 90 s in a bead beater (Bertin Technologies SAS, Paris, France). Then, the homogenate was transferred to a new tube, and protein concentration was calculated using a bicinchoninic assay (BCA, Thermo Fisher Scientific, Waltham, MA, USA). Fifty µg of protein was precipitated overnight, using 1:4 volume of cold (−20 °C) acetone. After overnight precipitation, the protein was pelleted by centrifugation at 13,500 rpm for 15 min at 4 °C. Subsequently, protein pellets were dried and dissolved in 8 M urea containing 10 mM dithiothreitol (DTT) and incubated at 37 °C for 1 h for disulfide bond reduction. Next, cysteines were alkylated by incubating the sample with an alkylating reagent mix consisting of 97.5% acetonitrile, 0.5% triethylphosphine, and 2% of iodoethanol for one h in the dark at room temperature. The samples were dried in a vacuum centrifuge. A trypsin/LysC combination (Promega, Madison, WI, USA) was used for sample digestion. The manufactured trypsin/LysC vial was dissolved in 400 µL of 50 mM ammonium bicarbonate, and 80 µL of the enzyme was added to each sample. Digestion was performed at high pressure using a Barocycler (50 °C, 60 cycles, 20,000 psi, Pressure Biosciences, Easton, MA, USA). Digested peptides were desalted using MicroSpin columns (C18 silica, The Nest Group Inc., Ipswich, MA, USA). Purified peptides were heated in a vacuum centrifuge and stored at −80 °C until analysis. Purified peptides were resuspended in 3% acetonitrile/0.1% formic acid to a final concentration of 1 µg/µL and 1 µL was used for LC-MS/MS analysis.