Proteomic Analysis of SOD1 and EfnA5 in ALS

Lumbar spinal cord samples were obtained from SOD1^WT, SOD1^G93A EfnA5^+/+ and SOD1^G93A EfnA5^+/− mice at 130 days of age after cervical dislocation, and were lysed by addition of lysis buffer containing 6 M guanidine hydrochloride, 10 mM Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), 40 mM 2-chloroacetamide and 100 mM triethylammonium bicarbonate (TEAB). Samples were then sonicated, heated at 95 °C for 10 min and centrifuged at 20000 g for 30 min at 4 °C, and the supernatant was collected for further analysis. Protein concentration was determined using the Qubit Fluorometric Quantification Kit (Thermo Scientific). Protein digestion was performed with a filter-aided sample preparation (FASP) protocol and analysed by LC-MS/MS as previously described [4 (link)]. Proteins were identified using MaxQuant 1.5.2.8 and the Mus musculus reference proteome from UniProt (downloaded 12th March 2018). A false discovery rate (FDR) of 1% was used for peptide and protein identification (total protein IDs after exclusion of contaminants: 5525) and protein quantification was performed with the MaxLFQ algorithm [8 (link)]. Quantitative analysis of the data was performed with Perseus 1.5.2.6 [40 (link)]. Proteins with valid values in at least three samples per group were used for the quantitative comparison (4065 proteins).