Neurite versus whole cell proteomics:

Boyden chambers were washed with cold PBS and neurites were scraped from the underside of the chamber. The top of the chamber was subsequently scraped as the whole cell fraction. Fractionated cells were pelleted, PBS removed, and cells flash frozen before proceeding to SP3/benzonase protocol as described (18). Briefly, samples were resuspended in SP3 buffer (50 mM Tris-HCI [pH = 8.0], 50 mM NaCl, 1% SDS, 1% Triton X-100, 1% NP-40, 1% tween 20, 1% glycerol, 1% sodium deoxycholate (w/v), 5 mM EDTA [pH = 8.0], 5mM dithiothreitol (DTT), 5 KU benzonase, and 1× complete protease inhibitor (Roche 05892970001)), then shaken at 1200 rpm, 65°C for 30 min. Cell lysates were alkylated using 500 mM iodoacetamide (IAA) in the dark and then reduced by 500 mM DTT. Alkylated proteins were captured by hydrophilic magnetic beads using a KingFisher APEX robot, followed by a trypsin/LysC digestion in 50 mM ammonium bicarbonate overnight at 37°C and 1200 rpm. Beads were removed using KingFisher APEX and digested peptides were lyophilized and reconstituted to 0.5 mg/mL with MS loading buffer (2% acetonitrile, 0.1% formic acid). The peptides were analyzed on an Orbitrap Eclipse mass spectrometer, coupled with FAIMS interface using data-independent acquisition (DIA) approach with scan range 400–1,000 m/z, and 24 isolation windows (8 m/z per window). The RAW data files were database searched against UniProtKB Human Database containing 20,435 reviewed entries using Spectronaut 19 with label free or SILAC pipeline. Carbamidomethyl on cysteine was used as fixed modification, and acetylation on protein N-terminus and oxidation on methionine were used as variable modifications. The false discovery rate of protein and peptide level were set at 1%, and cross-run medium normalization was enabled.