Triethylammonium bicarbonate teab

A three-part fractionation was used to decrease sample complexity prior to tryptic digestion and LC-MS analysis. As shown in Supplementary Figure S1, frozen brain hemisphere was homogenized in modified phosphate buffered saline (NaCl concentration = 1M) with Halt Protease and Phosphatase Inhibitor Cocktail (PPIC) on ice in 10 s bursts. Following centrifugation at 20,000 × g at 4°C for 10 min the supernatant was mixed with chilled methanol (MeOH) for protein precipitation, while the pelleted material was saved and stored at −80°C as the “membrane” fraction for future use. The supernatants were incubated with chilled MeOH on ice for 30 min followed by centrifugation. All materials were then re-suspended in 20 mM triethylammonium bicarbonate (TEAB), pH 8.0 (Sigma Aldrich), 0.5% w/v sodium deoxycholate (SDC) via bath sonication. A process aliquot was taken to determine protein concentration using the bicinchoninic acid (BCA) assay (Pierce) and verified by SDS-PAGE and staining with Sypro Ruby. Samples were then prepared for tryptic digestion and labeling with Tandem Mass Tag (TMT) reagents (Thermo Fisher Scientific), 50 μg of each sample was then precipitated in four volumes of chilled acetone to concentrate and de-salt the material.

5 mM dithiothreitol (Sigma) was used to reduce the protein solution at 56 °C. 30 min later, at room temperature, 11 mM iodoacetamide (Sigma) was added to alkylate the solution for 15 min in the dark. Triethylammonium bicarbonate (TEAB) (100 mM) (Sigma) was used to dilute the urea concentration of the protein sample to less than 2 M. For the first digestion overnight, trypsin (Promega, USA) was added to the sample (mass ratio, 1:50 trypsin:protein). For the next 4 h, a second digestion was performed (mass ratio, 1:100 trypsin:protein).

Aliquots with 100 mg of proteins from each sample were added to 100 ml of 200 mM triethyl ammonium bicarbonate TEAB (Sigma-Aldrich, St. Louis, MO). Reduction was performed by adding 5 ml of 200 mM tris (2-carboxyethyl) phosphine TCEP (Sigma-Aldrich, St. Louis, MO) to each replicate and incubating for 1 h at 55°C. Alkylation was carried out by adding 5 ml of 375 mM iodoacetamide (Bio-Rad Laboratories, Hercules, CA) to each sample and incubating for 30 min at room temperature. After alkylation, 1 ml of pre-chilled acetone was added and precipitation was allowed to proceed for 3 h at 20°C. Acetone-precipitated protein pellets were suspended in 100 ml of 200 mM TEAB and digested overnight at 37°C with 2.5 μg of sequencing grade modified trypsin (Promega Corp., Madison, WI) as previously described [13 (link),14 (link)].