Shotgun proteomics

Secretome analysis of primary neuronal cultures was performed using the high-performance secretome protein enrichment with click sugars" (hiSPECS) method, described in detail previously (Tüshaus et al, 2020). In brief, neurons were cultured for 48 h (DIV 5-7) in the presence of 50 µM ManNAz (#88904, ThermoFisher), cultivation media was filtered through 0.45 µm spin columns (Sigma-Aldrich, CLS8163). Glycoproteins were enriched using ConA agarose beads (Sigma, C7555) and clicked to magnetic DBCO beads (Jena Bioscience, CLK-1037). Peptides were released from the beads by on-bead tryptic digest and desalted using the stage tip protocol (Rappsilber et al, 2003). Finally, peptides were dissolved in a total volume of 20 µL (18 µL 0.1% FA with 2 µL iRT peptides (1:10, Ki-3002-1, Biognosys) of which 8 µL were injected per LC-MS/MS run. The CSF was isolated from iRhom1-/- mice and wildtype littermates at the age of 3 months (N=4) following the procedure described by (Lim et al, 2018; Liu & Duff, 2008). Mice were anesthetized with a cocktail of medetomidine, midazolam and fentanyl (0.05/5/0.5 mg/kg i.p.). Next, the mice were positioned into a stereotaxic frame, the cisterna magna was exposed and a glass capillary was used to puncture the dura and to harvest the CSF by applying slight negative pressure. The blood-free CSF samples were cleared by centrifugation at 3000 g for 20 min and snap-frozen in liquid nitrogen. 5 µL CSF was used for in-solution digestion including sodium deoxycholate as described by (Pigoni et al., 2016). All mass spectrometry measurements were conducted on an EASY‐nLC 1200 UHPLC system (Thermo Fisher Scientific) connected to a Q-Exactive™ HF Hybrid Quadrupole‐Orbitrap™ mass spectrometer (Thermo Fisher Scientific) as described (Tüshaus et al, 2021). Peptides were separated on self-packed 30 cm long C18 columns (ReproSil‐Pur 120 C18‐AQ resin, Dr. Maisch GmbH) with a diameter of 75 µm and a 120 min gradient (Tüshaus et al., 2020).