Proteomics analysis of myelin from GLTP KO mouse brains

Version 1

Tryptic in solution digestion of the purified myelin fraction was performed according to the filter-aided sample preparation (FASP) protocol (Erwig et al., 2019) originally described by Manza et al., 2005; followed by LC-MS-analysis. In brief, purified myelin fractions corresponding to 10 µg myelin protein were dissolved and lysed in lysis buffer (1% ASB-14, 7 M urea, 2 M thiourea. 10 mM DTT 0.1 M Tris pH 8.5). Homogenised samples were diluted with lysis buffer containing 2% CHAPS to reduce ASB-14 concentration below 0.1%. Subsequent steps were carried out in centrifugal filter units (30 kDa MWCO Vivacon® 500 spin filters, Sartorius). After depletion of detergents, protein samples were reduced by 10 mM dithiothreitol (DTT) and alkylated with iodoacetamide followed by a buffer exchange (50 mM acetonitrile bicarbonate (ABC)/10% acetonitrile) in which the samples were enzymatically digested overnight at 37°C with 400 ng trypsin (Promega). Peptides were recovered by an initial spin, followed by two rounds of centrifugation with 50 mM ABC. Subsequently, 1% trifluoroacetic acid (TFA) was added to the pooled flow-throughs before mixing peptides with a ratio 1:1 of 10% acetonitrile in 1% TFA. Peptides were desalted by custom-made C18 tips (Rappsilber et al., 2003). Samples were reconstituted in 0.1% (FA) and separated using an Easy nLC1200 liquid chromatograph (Thermo Scientific) followed by peptide detection on a Q Exactive HF mass spectrometer (Thermo Scientific). Separation was carried out on 75 µm × 15 cm custom-made fused silica capillary packed with C18AQ resin (Reprosil-PUR 120, 1.9 µm, Dr. Maisch) with a 140 min acetonitrile gradient in 0.1% FA at a flow rate of 400 nl/min (3–6 % ACN gradient for 2 min, 6–30% ACN gradient for 90 min, 30–44% ACN gradient for 20 min, 44–75% ACN gradient for 10 min, 75–100% ACN gradient for 5 min). Peptides were ionized using a Nanospray Flex Ion Source (Thermo Scientific). Peptides were identified in full MS / ddMS² (Top15) mode, dynamic exclusion was enabled for 20 s and identifications with an unassigned charge or charges of one or >8 were rejected. MS1 resolution was set to 120,000 with a scan range of 300–1700 m/z, MS2 to 15,000 and AGC target of 3e6.