Dystrophin protein, as well as myomesin and filamin c, were quantitated in different combination mixtures of muscle extracts from DMD and age matched healthy donors at ratios leading to 0, 5, 10, 25, 50, 75, 90, 95 and 100% of dystrophin relative to normal. Each final mixture contained 50 μg of total muscle protein and was spiked with 30 μg of 13C6-Lysine labeled SILAC mouse muscle extract. Gel bands encompassing dystrophin protein and other muscle specific proteins was excised and processed for mass spectrometry analysis as described in Methods. Transition ion intensities obtained at the MS/MS level for the targeted labeled and unlabeled peptide pairs generated from the spike-in standard and endogenous human dystrophin were used to determine the relative amount of dystrophin and other muscle proteins (myomesin and filamin c). Standard deviation at each data point represents average ratios obtained from labeled and unlabeled peptide pairs: 5 pairs for dystrophin protein (QAPIGGDFPAVQK, VLSQIDVAQK, IFLTEQPLEGLEK, TLNATGEEIIQQSSK, VHALNNVNK); 3 pairs for filamin c (SPFVVNVAPPLDLSK, EVGEHVVSVRK, HIGISFTPK) and two pairs for myomesin (VSEPVAALDPAEK, VLGGLPDWTIQEGK). These peptides have 100% sequence homology between human and mouse and were used for ratio measurements. As expected, dystrophin protein increased with increasing relative amount of normal muscle extract while myomesin and filamin c remained unchanged.
