Shotgun proteomics (human)

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.