Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal cells differentiation

N1E-115 cells were seeded two days before sample preparation, and cultured in fresh medium containing 2% fetal bovine serum for 12 h before sample preparation. Cells were washed with serum-free medium for three times, and cultured in fresh serum-free DMEM with or without 50 μM GM6001 for 9 h. The conditioned media were collected and centrifuged to remove cell debris, and concentrated up to ~45-fold. Concentrated media were acidified by acetate acidic buffer, and oxidized by NaIO4. Proteins were precipitated with methanol and chloroform, reduced, alkylated, and digested by trypsin. Digested peptides were purified using C18 column, concentrated, diluted with coupling buffer (Biorad), and incubated with Aff-Gel Hydrazide Gel (Biorad). N-glycosylated peptides were eluted and labeled with 18O by treatment with PNGase F (NEB). Eluted peptides were labeled with CH2O (light, DMSO-treated samples) or 13CD2O (heavy, GM6001-treated samples). Each sample was divided into two parts and subjected to LC-MS/MS analysis respectively.