Shotgun proteomics

Microglia isolated by MACS from WT and Myd88-/- mouse pups were seeded at a density of 1×106 cells per 60-mm dish in DMEM/FCS/L929 medium. After 2 DIV, microglia were washed with warm DMEM/pyruvate medium and treated with 20 µg/mL myelin debris (or HEPES control) in 2 mL of TIC medium for 4 h. After treatment, the cells were washed with DMEM/pyruvate medium, and incubated with 4 mL of DMEM containing 0.2% BSA for 16 h. The cells in each dish were washed with 2 mL of cold PBS on ice, and lysed with 200 µL of cell lysis buffer (Cell Signaling Technology, 9803) containing 1 mM PMSF for 5 min on ice. The cells were detached using cell scrapers, collected and stored at -80 °C. The mass spectrometry label-free quantification (LFQ) was performed for the cell lysate of cultured microglia (triplicates or quadruplicates of each condition). A protein amount of 15 µg of each cell lysate was subjected to proteolytic digestion with 0.3 µg LysC (Promega) and 0.15 µg trypsin (Promega) using the filter assisted sample preparation (FASP) protocol with 30 kDa Vivacon spin filters (Sartorius). Proteolytic peptides were desalted by stop and go extraction with C18 tips. The purified peptides were dried by vacuum centrifugation. Samples were dissolved in 20 µL 0.1% formic acid. Peptides were analysed on an Easy nLC 1200 nanoHPLC (Thermo Scientific) which was coupled online via a Nanospray Flex Ion Source (Thermo Sientific) equipped with a PRSO-V1 column oven (Sonation) to a Q-Exactive HF mass spectrometer (Thermo Scientific). An amount of 1.3 µg of peptides was separated on an in-house packed C18 column (30 cm x 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH) using a binary gradient of water (A) and acetonitrile (B) supplemented with 0.1% formic acid (0 min., 2% B; 3:30 min., 5% B; 137:30 min., 25% B; 168:30 min., 35% B; 182:30 min., 60% B) at 50°C column temperature with a flow rate of 250 nL/min. A data-dependent acquisition method was used. Full MS scans were acquired at a resolution of 120,000 (m/z range: 300-1400, AGC target: 3E+6). The 15 most intense peptide ions per full MS scan were selected for peptide fragmentation (resolution: 15,000, isolation width: 1.6 m/z, AGC target: 1E+5, NCE: 26%). A dynamic exclusion of 120 s was used for peptide fragmentation.