Cp02 automated dry pulverizer

Frozen tissue samples from heart and skeletal muscle were transferred into prechilled tubes and cryo-pulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) according to the manufacturer's instructions. Powdered tissue was lysed in 8 M urea/0.5 M NH₄HCO₃ by ultrasonicating (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) was used for protein quantification. Thirty micrograms of protein were digested with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h and subsequently with modified porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37°C.

Frozen lung tissue samples were placed into precooled tubes and cryopulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) with an impact level of 5 according to the manufacturer’s instructions. Tissue lysis was performed in 8 M urea/0.5 M NH₄HCO₃ with ultrasonication (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Total protein concentration was quantified using a Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Fifty micrograms of protein were digested sequentially, firstly with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h and, subsequently, with modified porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C.

Frozen liver tissue samples were transferred into prechilled tubes and cryo-pulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) using an impact level of 3 according to the manufacturer's instructions. Powdered tissue was lysed in 8 M urea/0.5 M ammonium bicarbonate (Roche Diagnostics, Mannheim, Germany) by ultrasonication (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA) was used for protein quantification. 20 μL of lysate containing 20 μg of protein were processed for digestion. Disulfide bonds were reduced with 45 mM dithiothreitol/20 mM tris(2-carboxyethyl) phosphine (30 min, 56 °C). Reduced cysteine side chains were alkylated by adding 100 mM iodoacetamide (30 min, room temperature), followed by quenching the remaining iodoacetamide with dithiothreitol (90 mM, 15 min, room temperature). Sequential 2-step digestion was performed, firstly with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h (1:50 enzyme to protein ratio) and subsequently with modified porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C (1:50 enzyme to protein ratio). After digestion, samples were dried before analysis using a vacuum centrifuge.