Shotgun proteomics

Blood samples (20 ml) from clinically affected homozygous Npc1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll gradient (800 x g for 15 min, GE Healthcare). Potential residues of red cells were eliminated by incubating cell pellets with ACK lysis buffer (Gibco) for 3 min at RT. Lysis buffer was quenched with 40 ml of washing buffer and cells were centrifuged at 300 x g for 7 min. Cell pellets were resuspended and plated in macrophage complete medium (RPMI1640/10% FBS/1% PenStrep/1X Pyruvate/1X NEAA) supplemented with 50 ng/ml hM-CSF (Thermo Scientific). After 48 h, 50 ng/ml of fresh hM-CSF was re-added. At 5DIV, media have been discarded and adherent cells have been washed once in PBS, incubated for 3 min at RT with Versene (Lonza) and scraped in 5 ml macrophage complete medium for further analysis. Human macrophages (7 NPC patients and 3 healthy controls) were lysed in 200 µl of STET lysis buffer (50 mM Tris/150 mM NaCl/2 mM EDTA/1% Triton X-100, pH 7.5) and incubated 15 min on ice 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 fresh protein LoBind tube (Eppendorf) and the protein concentration was estimated using the Pierce 660 nm protein assay (ThermoFisher Scientific). A protein amount of 15 µg was subjected to tryptic protein digestion using the filter aided sample preparation protocol (FASP) using Vivacon spin filters with a 30 kDa cut-off (Sartorius). Briefly, proteins were reduced with 20 mM dithiothreitol and free cysteine 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). After vacuum centrifugation, peptides were dissolved in 2 µl 0.1% formic acid (Biosolve) and indexed retention time peptides were added (iRT Kit, Biognosys). For label free protein quantification (LFQ), peptides were analysed 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. Data dependent acquisition (DDA) was used for LFQ. 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.

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 SP3 protocol (Hughes et al. 2018). 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 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 30 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). The maximum ion trapping time was set to “auto”. A stepped normalized collision energy of 26% ± 2.6% was used for fragmentation.

he microglia enriched fractions from MACS sorting were lysed in 200µl of STET lysis buffer (50mM Tris, 150mM NaCl, 2mM EDTA, 1% Triton X-100, pH 7.5) and incubated 15 min on ice with intermediate vortexing. The samples were centrifuged for 5min at 16,000xg at 4°C to remove cell debris and undissolved material. The supernatant was transferred to a fresh protein LoBind tube (Eppendorf) and the protein concentration was estimated using the Pierce 660nm protein assay (ThermoFisher Scientific). A protein amount of 15µg was subjected to tryptic protein digestion using the filter aided sample preparation protocol (FASP) using Vivacon spin filters with a 30kDa cut-off (Sartorius). Briefly, proteins were reduced with 20mM dithiothreitol and free cysteine residues were alkylated with 50mM iodoacetamide (Sigma Aldrich). After the urea washing steps, proteins were digested with 0.3µg LysC (Promega) for 16h at 37°C followed by a second digestion step with 0.15µg trypsin (Promega) for 4h 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) [83]. After vacuum centrifugation, peptides were dissolved in 20 µl 0.1% formic acid (Biosolve). For label free protein quantification (LFQ), peptides were analysed 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 1.3µg of peptides was separated on in-house packed C18 columns (30cm x 75µm 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. Data dependent acquisition (DDA) was used for LFQ. 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.