Shotgun proteomics

Corpus callosum dissections were lysed in 300 µL STET lysis buffer (1% (v/v) Triton X-100, 150 mM NaCl, 2 mM EDTA, 50 mM TrisHCl pH 7.5) with a Precellys Evolution homogenizer (Bertin, Germany) using 0.5 mL soft tissue homogenization kit CK14 applying two cycles of 30 s with a speed of 6500rpm. After 15 min incubation on ice, samples were centrifuged at 16,000×g for 15 min to remove undissolved material and cell debris. The supernatant was transferred to a fresh protein lobind tube (Eppendorf, Germany). The protein concentration of the lysates was estimated using the Pierce 660 nm assay (ThermoFisher Scientific, US). A protein amount of 20 µg per sample was subjected to tryptic digestion. First, 100 mM MgCl2 was added to a final concentration of 10 mM and DNA was digested with 25 units Benzonase (Sigma Aldrich, US) for 30 min at 37°C. Proteins were reduced at 37°C for 30 min with 15 mM dithiothreitol (DTT) followed by cysteine alkylation with 60 mM iodoacetamide (IAA) for 30 min at 20 °C. Excess of IAA was removed by adding DTT. Detergent removal and subsequent digestion with 0.25 µg LysC and 0.25 µg trypsin (Promega, Germany) was performed using the single-pot, solid-phase-enhanced sample preparation as previously described (Hughes et al., 2019). Proteolytic peptides were dried by vacuum centrifugation and dissolved in 20 µl 0.1% (v/v) formic acid. 350 ng of peptides were separated on a nanoElute nanoHPLC system (Bruker, Germany) using a 5 mm trapping column (Thermo Scientific, US) and an in-house packed C18 analytical column (30 cm × 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH). Peptides were separated with a binary gradient of water and acetonitrile (B) containing 0.1% formic acid at flow rate of 300 nL/min (0 min, 2% B; 5 min, 5% B; 92 min, 24% B; 112 min, 35% B; 121 min, 60% B) and a column temperature of 50°C. The nanoHPLC was online coupled to a TimsTOF pro mass spectrometer (Bruker, Germany) with a CaptiveSpray ion source (Bruker, Germany). A standard Data Dependent Acquisition Parallel Accumulation–Serial Fragmentation (DDA-PASEF) method with a cycle time of 1.1 s was used for spectrum acquisition (Meier et al., 2018). Briefly, ion accumulation and separation using Trapped Ion Mobility Spectrometry (TIMS) was set to a ramp time of 100 ms. One scan cycle included one TIMS full MS scan and 10 PASEF peptide fragmentation scans. The m/z scan range was set to 100-1700 for both, MS and MS/MS scans. For lipidomics, lipid species and subspecies are annotated according to their molecular composition as described previously and lipid identifiers are provided (Aimo et al., 2015) (Table S3). One μL of homogenized and diluted brain tissue was analyzed using Shotgun lipidomics platform by Lipotype GmbH (Dresden, Germany), as described previously (Surma et al., 2015).