Incorporation and mixing check

The efficiency of SILAC labeling was determined by LC-MS/MS measurement of Lys4- and Lys8-labeled samples. For that, 10 μg of each sample was separately digested with endoproteinase Lys-C, purified by StageTips (see below), and measured by LC-MS/MS. In all cases, the labeling efficiencies of Lys4 or Lys8 were ≥94%. Before the mixing of labeled samples for SILAC experiments, 20 μg of each differentially labeled sample was premixed in equal protein amounts determined by Bradford assay, digested with endoproteinase Lys-C, and measured by LC-MS/MS. Median of evidence SILAC ratios was used as a correction factor for mixing the samples to be used in main SILAC experiments.