Phosphoproteomics / Bottom-up proteomics (mouse)

Phosphoproteomics: starting from the protein extracts from the global proteomics experiments, proteases inhibitors (Sigma, P8340) and phosphatases inhibitors (final concentration in Na3VO4 = 1 mM) were added to all samples. Protein concentration was determined using RC-DC assay (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. 250 µg of proteins for each sample were reduced and alkylated prior to an in-house optimized single-pot, solid-phase-enhanced sample preparation (SP3) protocol. Briefly, beads A (Sera-Mag Speed beads, Fisher Scientific, Germany, 45152105050250) and beads B (Sera-Mag Speed beads, Fisher Scientific, Germany, 65152105050250) were combined (ratio 1:1) and, after 3 washing steps with H2O, were added to the samples (ratio beads:protein of 10:1 for each type of beads, meaning a 20:1 ratio for the combination of beads). After inducing protein binding to the beads with 100% ACN for 18 minutes, the beads/proteins mixtures were washed twice with 80% EtOH and once with 100% ACN before being resuspended in 95 µL NH4HCO3 prior to overnight on-beads digestion (enzyme:protein ratio of 1:20) at 1 000 rpm at 37°C using modified porcine trypsin/lys-C (Mass Spec Grade mix, Promega, Madison, USA). Digestion was stopped using TFA (final pH < 2). Recovered peptides were resuspended in 170 µL of 80% ACN, 0.1% TFA and phosphomix I light (Sigma Aldrich) was added to each sample (ratio peptide (µg)/mix(fmol) = 1.6). Phosphopeptide enrichment was performed on 5 µL phase Fe(III)-NTA cartridges on an AssayMAP Bravo platform following an IMAC protocol. After the enrichment, FA was added to each sample as well as phosphomix I heavy (Sigma Aldrich) (ratio peptide (µg)/mix(fmol) = 1.6). Dried phosphopeptides were resuspended in 40 µL H2O, 2% ACN, 0.1% FA. Sample preparation steps for C9 & FUS mouse models were identical to those previously described for SOD1 & TDP-43, except that proteins were extracted from new tissue samples just before the ph-proteomics experiment. Nano-LC-MS/MS analyses were performed on a nanoAcquity UPLC devise (Waters) coupled to a Q-Exactive HF-X mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a Nanospray Flex™ ion source. Phosphopeptides were separated at 400 nL/min with 105 min gradient of ACN. The system was operated in DDA mode and the ten most abundant ions were selected on each MS spectrum for further isolation and HCD, excluding monocharged and unassigned ions.