In-Solution Protein Digestion and LC-MS/MS Analysis

For in-solution digests, 6 μg of protein was reduced with 5 mM dithiothreitol (DTT) (AppliChem) for 20 min at RT and subsequently alkylated with iodoacetamide (Sigma-Aldrich) for 20 min at 37°C. Trypsin (Thermo Scientific) was added in a 1:50 enzyme–protein ratio and digested overnight at 37°C. Samples separated via SDS–PAGE were stained by Coomassie. Each gel lane was cut into 20 pieces and prepared for LC/MS analysis as described (Hecht et al., 2019 (link)). LC/MS and bioinformatical analysis were carried out as described above, with the exception of shortening the LC gradient to 65 min in total. Employing an LTQ Orbitrap Elite system (Thermo Fisher Scientific) online coupled to an U3000 RSLCnano (Thermo Fisher Scientific), samples were analyzed as described (Mohr et al., 2015 (link)), with modifications (Hecht et al., 2019 (link)) and picking the 20 most intense ions from the survey scan for CID fragmentation. Singly charged ions were rejected and m/z of fragmented ions were excluded from fragmentation for 60 s. MS2 spectra were acquired employing the LIT at rapid scan speeds.