Shotgun proteomics

Nine patients per group were treated with either acitretin or vehicle control. Cerebrospinal fluid was collected before (baseline value) and after treatment. A volume of 5 µL of CSF per sample was subjected to proteolytic digestion in 50 mM ammonium bicarbonate with 0.1% sodium deoxycholate (Sigma Aldrich, Germany) as previously described (Pigoni et al., 2016). Briefly, protein disulfide bonds were reduced with dithiothreitol and sulfhydryl residues were alkylated using iodoacetamide. Proteins were digested using 0.1 µg LysC (Promega) and 0.1 µg trypsin (Promega). Deoxycholate was precipitated by acidification and removed by centrifugation at 16,000 rcf and 4°C for 10 min. Proteolytic peptides were desalted by stop and go extraction (STAGE) with C18 tips (Rappsilber et al., 2003). The purified peptides were dried by vacuum centrifugation. Samples were dissolved in 20 µL 0.1% formic acid. Samples were separated on a nanoLC system (EASY-nLC 1000, Proxeon – part of Thermo Scientific, US; PRSO-V1 column oven: Sonation, Germany) using an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH, Germany) with a binary gradient of water (A) and acetonitrile (B) containing 0.1% formic acid at 50°C column temperature and a flow of 250 nl/min (0 min, 2% B; 3:30 min, 5% B; 137:30 min, 25% B; 168:30 min, 35% B; 182:30 min, 60% B). The nanoLC was coupled online via a nanospray flex ion source (Proxeon – part of Thermo Scientific, US) to a Q-Exactive mass spectrometer (Thermo Scientific, US). Full MS spectra were acquired at a resolution of 70,000. The ten most intense ions exceeding an intensity of 1.5×104 were chosen for collision induced dissociation and spectra were acquired at a resolution of 17,500. The dynamic exclusion for peptide fragmentation was set to 120 s.