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Schematic of the workflow. Tissue lysates from each of four mice aged 5 or 26 months were analyzed in separate experiments and mixed each time with an identical standard derived from SILAC mice aged 12 months. Proteins were digested using the filter-aided sample preparation method (FASP) and fractionated before analysis by high resolution mass spectrometry. Proteins were quantified by dividing the individual SILAC peptide ratios of the proteomes to be compared (“ratio of ratios”).
