In each SILAC experiment, differently labeled protein extracts were mixed in equal amounts corrected by the ratios determined by measuring mixing checks (see below) to a total of 12 mg. Proteins were reduced using 1 mM dithiothreitol (DTT) for 1 hour and subsequently alkylated with 5.5 mM iodoacetamide for 1 hour. One-half of the protein mixture was diluted with four volumes of 62.5 mM tris (pH 8.0) and 12.5 mM CaCl2 and digested with chymotrypsin (1:120, w/w) overnight at room temperature (RT). The other half was predigested with endoproteinase Lys-C (1:100, w/w) for 3 hours, then diluted with four volumes of 62.5 mM tris (pH 8.0), and supplemented with endoproteinase Lys-C (1:100, w/w) for overnight digestion at RT. The reaction was stopped by acidification with trifluoroacetic acid (TFA) to pH 2. An aliquot of 10 μg was purified by StageTips (see below), and 2 μg was used for direct proteome measurement with 230-min LC gradient. An additional aliquot of at least 100 μg intended for further proteome measurements was stored at −80°C.

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