Shotgun proteomics

After washing the primary cells with 1x PBS, cell-type specific growth media containing serum supplements with 50 µM of ManNAz (Thermo) was added for 48h. Afterwards, conditioned media was collected and filtered through Spin-X 0.45 µM cellulose acetate centrifuge tube filter (#8163, Costar) and stored at -20°C in protein Lobind tubes until further usage. Glycoprotein enrichment was performed using 60 µL Concanavalin A (ConA) bead slurry per sample (Sigma). ConA beads were washed twice with 1 mL of binding buffer (5 mM MgCl2, 5 mM MnCl2, 5 mM CaCl2, 0.5 M NaCl, in 20 mM TrisHCL pH 7.5) before use. The conditioned medium was incubated with the ConA beads for 2h in an overhead rotator at room temperature. The ConA beads were pelleted by centrifugation (2000 g, 1 min) and the supernatant containing unbound proteins was discarded. The beads were washed three times with 1 mL binding buffer before adding 500 µL of elution buffer (500 mM Methyl-alpha-D-mannopyranoside, 10 mM EDTA in 20 mM TrisHCl pH 7.5) and rotating over-head for 30 min at RT. The eluate was filtered through pierce spin columns (Thermo) to remove remaining ConA beads and then the filtrate was transferred to a 1.5 mL protein Lobind tube. The elution step was repeated with another 500 µL elution buffer and combined with the first eluate. 50 µL of magnetic DBCO beads (Jena bioscience) were washed twice with mass spec grade water and added to the eluate. Sodium deoxycholate (SDC) was added to a final concentration of 0.1% (w/v) (except otherwise noted).The click reaction was performed overnight at 4°C on an Eppendorf Thermomixer R shaker at 1200 rpm to covalently couple metabolically labeled glycoproteins to the magnetic beads. The next day, beads were washed three times with 1 mL 1% SDS buffer (100mM TrisHCl pH 8.5, 1% SDS, 250 mM NaCl), three times with 1 mL 8 M UREA buffer (8 M Urea in 100mM TrisHCl pH 8.5) and three times with 1 mL 20% (v/v) acetonitrile. Beads were retained with a magnetic rack. Beads were transferred to a new 1.5 mL low binding tube using 2 x 500 µL mass spec grade water and the supernatant was removed. Protein disulfide bonds were reduced in 50 µL of 10 mM DTT in 100 mM ABC for 30 min at 1200 rpm at 37°C. Afterwards, the supernatant was discarded. Alkylation of cysteines was performed using 50 µL of 55 mM IAA in 100mM ABC for 30 min at 1200 rpm and 20°C in the dark. The supernatant was discarded and beads were washed twice with 100 µL of 100 mM ABC. The protein digestion was performed by adding 0.2 µg LysC (Promega) in 50 µL of 100 mM ABC for 3h at 1200 rpm at 37°C followed by overnight trypsin digestion using 0.2 µg of trypsin (Promega) per sample in 100 mM ABC without 0.1% SDC. The supernatant containing the tryptic peptides was transferred to a 0.5 mL protein Lobind tube. Beads were washed twice with 100 mM ABC without 0.1% SDC and added to the same tube. Each sample was acidified with 50 µL of 8% FA and incubated for 20 min at 4°C. Precipitated SDC was removed by centrifugation at 18.000 g for 20 min at 4 °C. Peptides were cleaned up using C18 Stage tips. Dried peptides were resuspended in 18 µL 0.1% formic acid (FA) and 2 µL of 1:10 diluted iRT peptides (Biognosys) were spiked into the samples. CSF samples were processed as previously described (Pigoni et al. 2016). The LC-MS/MS analyses were performed on an EASY-nLC 1200 UHPLC system (Thermo) which was online coupled with a NanoFlex ion source equipped with a column oven (Sonation) to a Q Exactive™ HF Hybrid Quadrupol-Orbitrap™ mass spectrometer (Thermo). 8 µL per sample were injected. Peptides were separated on a 30 cm self-made C18 column (75 µm ID) packed with ReproSil-Pur 120 C18-AQ resin (1.9µm, Dr. Maisch). For peptide separation, a binary gradient of water and 80% acetonitrile (B) was applied for 120 min at a flow rate of 250 nL/min and a column temperature of 50 °C: 3% B 0 min; 6% B 2 min; 30% B 92 min; 44% B 112 min; 75% B 121 min. DDA was used with a full scan at 120,000 resolution and a scan range of 300 to 1400 m/z, AGC of 3x106 ions and a maximum IT of 50 ms. The top 15 most intense peptide ions were chosen for HCD fragmentation. An isolation window of 1.6 m/z, a maximum IT of 100 ms, AGC of 1x105 were applied and scans were performed with a resolution of 15,000. A dynamic exclusion of 120 s was used. DIA was performed using a MS1 full scan followed by 20 sequential DIA windows with variable width for peptide fragment ion spectra with an overlap of 1 m/z covering a scan range of 300 to 1400 m/z. Full scans were acquired with 120,000 resolution, AGC of 5x106 and a maximum IT time of 120 ms. Afterwards, 20 DIA windows were scanned with a resolution of 30,000 and an AGC of 3x106. The maximum IT for fragment ion spectra was set to auto to achieve optimal cycle times. The m/z windows were chosen based on the peptide density map of the DDA run of a representative iSPECS sample and optimized in a way that allowed the detection of 8 points per peak.