To obtain a deeper coverage of proteome samples, peptides from one replicate of each experiment were additionally separated offline using a high-pH reversed-phase peptide fractionation kit (catalog no. 84868, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, 50 μg of SILAC mixture was loaded onto the spin column, and peptides were eluted in the gradient of acetonitrile in nine fractions. The pH of 10 was maintained constant with 10 mM ammonium hydroxide instead of trimethylamine. Eluted fractions were concentrated by vacuum centrifugation and purified by StageTips (see below). Fractions were measured separately by LC-MS/MS using optimized LC gradients.

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