The excised FFPE brain regions were deparaffinized with n-heptane followed by ethanol washes and lysed in 100 mM triethyl ammonium bicarbonate (TEAB) and 1% w/w sodium deoxycholate (SDC) buffer, boiled at 95 °C for 60 min antigen retrieval. Samples were sonicated with a probe sonicator for 10 pulses to destroy DNA/RNA.
The excised fresh frozen brain regions were lysed in 100 mM TAB and 1% w/w SDC buffer, boiled at 95 °C for 5 min and sonicated with a probe sonicator for 10 pulses. For proteome analysis 20 µg for phosphopeptide enrichment (FF only) 200 µg of protein of each sample was taken and disulfide bonds reduced in the presence of 10 mM dithiotreitol (DTT) at 60 °C for 30 min. Cysteine residues were alkylated in presence of 20 mM iodoacetamide at 37 °C in the dark for 30 min and tryptic digestion (sequencing grade, Promega) was performed at a 100:1 protein to enzyme ration at 37 °C over night. Digestion was stopped and SDC precipitated by the addition of 1% v/v formic acid (FA). Samples were centrifuged at 16.000 g for 5 min and the supernatant was transferred into a new tube. Samples were dried in a vacuum centrifuge.
The excised fresh frozen brain regions were lysed in 100 mM TAB and 1% w/w SDC buffer, boiled at 95 °C for 5 min and sonicated with a probe sonicator for 10 pulses. For proteome analysis 20 µg for phosphopeptide enrichment (FF only) 200 µg of protein of each sample was taken and disulfide bonds reduced in the presence of 10 mM dithiotreitol (DTT) at 60 °C for 30 min. Cysteine residues were alkylated in presence of 20 mM iodoacetamide at 37 °C in the dark for 30 min and tryptic digestion (sequencing grade, Promega) was performed at a 100:1 protein to enzyme ration at 37 °C over night. Digestion was stopped and SDC precipitated by the addition of 1% v/v formic acid (FA). Samples were centrifuged at 16.000 g for 5 min and the supernatant was transferred into a new tube. Samples were dried in a vacuum centrifuge.