(A) Workflow for the identification of ADAM10 substrates in the secretome of primary cortical neurons comprising plating of neurons, lentiviral transduction, metabolic glycan labeling, purification and protein identification and quantification via mass spectrometry. Staining of primary cortical neurons for the neuron specific marker beta III tubulin and the nuclear stain DAPI followed by quantification of beta III tubulin staining supports that neuronal differentiation was unaffected by deletion of ADAM10. (B) Topology of all glycoproteins in the secretome. (C) Volcano plot of the quantitative comparison between the secretomes of wild type (wt) and Adam10 knockout neurons of all in at least 4 out of 5 experiments identified glycoproteins in the secretome of Adam10-/- and wt neurons. The p-value is depicted as negative decadic logarithm while the fold-change (Adam10-/-/wt) is depicted as log2 value. Significantly changed protein: p≤0,05 (-log10≥1,3). Substrates that are significantly reduced are marked red. Proteins which were validated with immunoblot have a yellow ring. Proteins which have been formerly described in literature as ADAM10 substrates are marked with a green cross. Hits whose p-values survived correction for multiple hypothesis testing yielded a p-value of at least 0.00135 as new significance niveau. (D) Topology of all significantly changed proteins in the secretome between Adam10-/- and wt neurons. (E) Percentage of all membrane proteins with type-I topology in the total secretome and among the significantly changed proteins upon Adam10 knockout. (F) Remaining shedding after Adam10 deletion in percent for members of selected protein families like the Neurexin (NRXN), Neuroligin (NLGN), receptor tyrosine phosphatase (PTPR), Slit and receptor tyrosine kinase domain (SLITRK) and the L1 family.