Comparative Proteomics of U251N Cells

To compare the proteomes of the U251N shCTRL and shCAPN2 cells, 10⁶ cells were collected and washed with DPBS. Proteins were extracted and desalted with S-Trap™ mini columns according to the manufacturer’s instructions (ProtiFi™). Proteins were digested with trypsin (V5111, Promega). Peptide concentrations were determined using Pierce™ BCA Protein Assay Kit. 25 µg of each sample were labelled with TMTpro™ 16plex Label Reagent Set (A44520, Thermo Scientific™) and pooled. 100 µg of the pooled sample were fractionated on a high pH liquid chromatography (HPLC, Agilent 1100 Series). 54 fractions were collected and concatenated to 18 samples which were submitted for LC–MS/MS analysis on a Velos Pro Orbitrap Elite™ (Thermo Scientific™) mass spectrometer coupled to an Easy nanoLC 1000 (Thermo Scientific™) with a 200 cm µPAC™ column (PharmaFluidics). Data were analyzed using MSFragger in Fragpipe 16.0 [51 (link)] and the UniProt human reference proteome database downloaded on the 14^th of June 2021 (20,845 protein entries including 245 potential contaminants). We allowed for two missed cleavages, a fragment and a precursor mass tolerance of 20 ppm each. A mass shift of 304.207146 Da was set for the N-term and lysine (K) representing the tandem mass tags (TMT), a mass shift of 57.0214560 Da for cysteine (C) representing carbamidomethylations and of 40.010600 Da for N-term acetylation.