Shotgun proteomics

Primary microglia were isolated from mouse brains (cerebrum) using MACS Technology (Miltenyi Biotec) according to manufacturer´s instructions and as previously described (Daria et al., 2017). Briefly, brain cerebrum was dissected, freed from meninges and dissociated by enzymatic digestion using a Neural Tissue Dissociation Kit P (Miltenyi Biotec) and subsequent mechanical dissociation using 3 fire-polished glass Pasteur pipettes of decreasing diameter. CD11b positive microglia were magnetically labelled using CD11b MicroBeads, loaded onto a MACS LS Column (Miltenyi Biotec) and subjected to magnetic separation, resulting in CD11b-enriched (microglia-enriched) and CD11b-depleted (microglia-depleted) fractions. Obtained microglia-enriched pellets were either washed twice with HBSS (Gibco) supplemented with 7mM HEPES, frozen in liquid nitrogen and stored at -80°C for biochemical or mass-spectrometry analysis or resuspended in microglial culturing media and used for phagocytosis assay as described below. Microglia-enriched pellets were lysed in 200 µL of STET lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% Triton, pH 7.5) at 4°C with intermediate vortexing. The samples were centrifuged for 5 min at 16,000 x g at 4°C to remove cell debris and undissolved material. The supernatant was transferred to a LoBind tube (Eppendorf) and the protein concentration estimated using the Pierce 660 nm protein assay (ThermoFisher Scientific). A protein amount of 15 µg was subjected to tryptic protein digestion applying the the filter aided sample preparation protocol (FASP) (Wisniewski, Zougman et al., 2009) using Vivacon spin filters with a 30 kDa cut-off (Sartorius). Briefly, proteins were reduced with 20 mM dithiothreitol and free cystein residues were alkylated with 50 mM iodoacetamide (Sigma Aldrich). After the urea washing steps, proteins were digested with 0.3 µg LysC (Promega) for 16 h at 37°C followed by a second digestion step with 0.15 µg trypsin (Promega) for 4 h at 37°C. The peptides were eluted into collection tubes and acidified with formic acid (Sigma Aldrich). Afterwards, proteolytic peptides were desalted by stop and go extraction (STAGE) with self-packed C18 tips (Empore C18 SPE, 3M) (Rappsilber, Ishihama et al., 2003). After vacuum centrifugation, peptides were dissolved in 20 µL 0.1% formic acid (Biosolve) and indexed retention time peptides were added (iRT Kit, Biognosys). For label free quantification (LFQ) of proteins, peptides were analyzed on an Easy nLC 1000 or 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 in-house packed C18 columns (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. For data dependent acquisition (DDA), 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. For data independent acquisition (DIA), one scan cycle included a full MS scan (m/z range: 300-1400; resolution: 120,000; AGC target: 5E+6 ions) and 25 MS/MS scans covering a range of 300-1,400 m/z with consecutive m/z windows (resolution: 30,000; AGC target: 3E+6 ions; Table EV1). The maximum ion trapping time was set to “auto”. A stepped normalized collision energy of 26% ± 2.6% was used for fragmentation. Microglia from APPPS1 mice were analyzed using DDA and DIA for method establishement. Microglia from APPPS1 and APP-KI mice were compared using DIA as DIA outperformed DDA.