Probe sonication

Brain tissue samples stored in 1.5 mL tubes were delivered to the Duke Proteomics Metabolomics Core Facility (n = 6 per genotype). 0.5% w/v ALS-1 surfactant in 50 mM ammonium bicarbonate (AmBic) was added to each sample at a volume of 10 uL/mg wet weight of tissue. Tissue homogenization and cell lysis was performed with probe sonication (Misonix) over three pulses at power level 3 for 5 seconds each with cooling on ice between pulses. A 5 uL aliquot of homogenate was diluted 25x in AmBic for determination of protein content by Bradford assay. Based on Bradford results, samples were 0.7 ± 0.2 mg protein / mg tissue. Following normalization (100 μg protein at 1 mg/mL protein in 0.5% ALS-1/AmBic), samples were reduced with 10 mM dithiothreitol (DTT) at 80◦ C with shaking for 15 minutes, alkylated with 20 mM iodoacetamide (IAA) at room temperature in the dark for 30 minutes, and digested with 2 μg sequencing grade modified trypsin (Promega) overnight at 37◦ C with shaking. Digestion was stopped with the addition of 12 μL 10/20/70 v/v/v TFA/MeCN/H2O and heating at 60◦ C for 2 hours. Diluted further with 1/2/97 v/v/v TFA/MeCN/H2O for a final digested protein concentration of 0.5 ug/uL. A pool of all samples (Study Pool QC, SPQC) was created from equal volume of each sample, and analyzed at regular intervals throughout the study to allow observation of any experimental drift.