Lipids were removed from synaptosome samples (n = 5 + 5 mice/genotype) as previously described (Gorski et al., 2020 (link)). Briefly, samples were incubated overnight at −20°C in five volumes ice-cold (−20°C) acetone and centrifuged twice at 1000 × g for 10 min at +4°C with an additional acetone wash in between. Pellets were air dried for 5 min and resuspended in freshly prepared 6.0 M urea/25 mM ammonium bicarbonate. Protein concentrations (μg/μl) were determined spectrophotometrically using the BCA protein assay kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions.
For proteome analysis, 5 μg of each sample was reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA), and the urea concentration was diluted to 1 M, followed by overnight digestion with trypsin (Promega Corporation, WI, USA) at +37°C. Peptides were desalted and concentrated by the STAGE-TIP method using a C18 resin disk (3 M Empore). Samples were eluted with 0.1% formic acid/60% acetonitrile, dried, and solubilized in 7 μl 0.1% formic acid prior to mass spectrometry analysis. Each peptide mixture was analyzed using an EASY-nLC system coupled to the QExactive Plus mass spectrometer (ThermoElectron, Bremen, Germany) equipped with the EASY Spray PepMap®RSLC column (C18, 2 μl, 100 Å, 75 μm x 25 cm) using a 120 min LC separation gradient.
For proteome analysis, 5 μg of each sample was reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA), and the urea concentration was diluted to 1 M, followed by overnight digestion with trypsin (Promega Corporation, WI, USA) at +37°C. Peptides were desalted and concentrated by the STAGE-TIP method using a C18 resin disk (3 M Empore). Samples were eluted with 0.1% formic acid/60% acetonitrile, dried, and solubilized in 7 μl 0.1% formic acid prior to mass spectrometry analysis. Each peptide mixture was analyzed using an EASY-nLC system coupled to the QExactive Plus mass spectrometer (ThermoElectron, Bremen, Germany) equipped with the EASY Spray PepMap®RSLC column (C18, 2 μl, 100 Å, 75 μm x 25 cm) using a 120 min LC separation gradient.
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