Reversed phase solid phase extraction cartridges

The protein extracts were processed following the filter-aided sample preparation protocol (23 (link)) with slight modifications. Briefly, protein extracts were applied on 10k filtration units (MilliporeSigma) and reduced with 1 mM DTT in the presence of 8 M urea and 50 mM HEPES. Upon centrifugation, proteins were alkylated with 5.5 mM iodoacetamide in the presence of 4 M urea and 50 mM HEPES and centrifuged again. Subsequently, proteins were washed once before digestion at 37°C for 4 h with Lys-C 1:50 w/w (Wako Pure Chemicals, Tokyo, Japan), both in the presence of 0.5 M urea and 50 mM HEPES. Finally, a 50-mM HEPES solution containing trypsin 1:25 (w/w) (Thermo Fisher Scientific) was added and samples were incubated overnight at 37°C. The peptides were collected by centrifugation of the filter-aided sample preparation filters, and the samples were desalted using polymeric Reversed Phase–Solid Phase Extraction cartridges (Phenomenex, Torrance, CA, USA). Peptide concentration was measured with DC Protein Assay and separate 500 and 50 μg aliquots of peptides/sample were lyophilized and set aside for phosphoproteomics and proteomics analysis, respectively.