Untargeted Proteomics (mouse)

Cells were collected and lysed in urea buffer (9 M Urea, 50 mM Tris pH 8, 150 mM NaCl, 1x Roche protease inhibitor cocktail) followed by short sonification. Samples were cleared by centrifugation and protein amounts were adapted. Protein reduction was performed with dithiothreitol (DTT; 5 mM final) for 25 min at 56°C and protein alkylation by the addition of iodoacetamide (14 mM final) for 30 min at room temperature. Protein mixtures were quenched with DTT and diluted 1:5 with 1 M Tris-Hcl, pH 8.2. For increased peptide recovery, proteins were digested at room temperature for 3 h with LysC (FUJIFILM, 2 µl/100 µg protein) before overnight tryptic digest at 37°C (0.5 µg/100µg protein). The following day, digestion was stopped with 10% TFA. To increase analysis depth, samples were pre-fractioned by a C18-SCX custom-made stage tip6. Fractions were eluted stepwise with increasing NH4AcO concentrations (20 mM to 500 mM) and desalted on a separate C18 stage tip. Desalted peptides were loaded on a custom-made 75 mm x 15 cm fused silicia capillary filled with C18-AQ resin (Reprosil Pur 120, 1.9 µm, Dr. Maisch HPLC) using an Easy-nLC1200 liquid chromatography. Samples were separated for 75 min with a 5-95% ACN gradient in 0.5% acetic acid using a Q Exactive HF mass spectrometer (Thermo Scientific).