Macrospin

An extended description of sample preparation for proteomics is in Appendix A Supplementary Materials & Methods. In short, tissues were pulverized and homogenized in lysis buffer containing 100 mM Tris–HCl pH7.5, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1% Triton X-100, cOmplete inhibitor cocktail (Roche) and PhosSTOP (Roche). Samples were lysed by polytron followed by ultrasonication. Proteins were precipitated by trichloroacetic acid, alkylated digested with modified trypsin (enzyme/protein ratio 1:50) overnight. Peptides were desalted using C18 reverse-phase spin columns (Macrospin, Harvard Apparatus) according to the manufacturer's instructions, dried under vacuum and stored at −20 °C until further use.
For TMT-labelling, 25 μg of peptides per sample were labeled with isobaric tandem mass tags (TMT10plex, Thermo Fisher Scientific) as described [28 (link)].

The dried peptide samples were subsequently labeled with isobaric tag (TMT 10-plex, Thermo Fisher Scientific) according to the manufacturer’s instructions. To control for ratio distortion during quantification, a peptide calibration mixture consisting of six digested standard proteins mixed in different amounts were added to each sample before TMT labeling as recently described^{60 (link)}. After pooling the TMT labeled peptide samples, peptides were again desalted on C18 reversed-phase spin columns according to the manufacturer’s instructions (Macrospin, Harvard Apparatus) and dried under vacuum. TMT-labeled peptides were fractionated by high-pH reversed phase separation using a XBridge Peptide BEH C18 column (3,5 µm, 130 Å, 1 mm × 150 mm, Waters) on an Agilent 1260 Infinity HPLC system. Peptides were loaded on column in buffer A (ammonium formate (20 mM, pH 10) in water) and eluted using a two-step linear gradient starting from 2% to 10% in 5 min and then to 50% (v/v) buffer B (90% acetonitrile/10% ammonium formate (20 mM, pH 10) over 55 min at a flow rate of 42 µl/min. The elution of peptides was monitored with a UV detector (215 nm, 254 nm). A total of 36 fractions were collected, pooled into 12 fractions using a post-concatenation strategy as previously described^{61 (link)}, dried under vacuum and subjected to LC-MS/MS analysis.