Fig. 1: Quantitative analysis of the neuronal surface using autoCSC.

a Cultured cortical neurons were collected every second day from 2 to 20 DIV and subjected to autoCSC surface labeling. Live cells were oxidized under mild conditions with sodium-meta-periodate and subsequently labeled with cell-impermeable biocytin-hydrazide. After cell lysis and tryptic digestion, the resulting peptides were subjected to automated processing on a liquid handling robot. Repeated aspiration through filter tips containing streptavidin resin captures biotinylated N-glycopeptides. After extensive washing, peptides were released using PNGase F, which catalyzes the cleavage of asparagine-linked glycans. Cleavage leaves a modification (deamidation) at asparagines that can be identified using high-resolution MS. Initially labeled and extracellular peptides are identified by presence of deamidated asparagines within the NXS/T glycosylation consensus sequence, indicating both surface localization and glycosylation site. DIA and targeted feature extraction were used to quantify surface-protein abundances across multiple conditions, providing quantitative surface abundance profiles across neuronal development in culture. b Qualitative overview of the neuronal surfaceome composition illustrated by intersections of quantified proteins for each time point. c Correlation of all quantified protein abundance values per DIV (median per time point). Source data are provided as a Source Data file.