Figure 1: Overview of the experimental design used to quantitate dystrophin protein in human skeletal muscle.
Total protein extracts from muscle biopsies (50 μg aliquots) were further fractionated on SDS-PAGE using 3-8% Tris-Acetate gel, which is suitable for high molecular mass proteins. Left panel shows the post-digestion spike-in approach where the area corresponding to dystrophin migration level (dashed blue lines) was serially sliced, in-gel digested by trypsin and spiked with stable isotope labeled standard peptides (see supplemental Figure S2 for peptide sequences and their location within the dystrophin protein) for subsequent LC-MS/MS analysis and peptide quantitation. In the Pre-digestion spike-in approach (right panel), 50 μg total muscle protein extract was spiked with 30 μg of 13C6-Lysine labeled SILAC mouse muscle protein extract. The mixture was then fractionated by SDS-PAGE and the area corresponding to dystrophin migration was excised, in-gel digested and the resulting peptides were analyzed by LC-MS/MS for identification and quantitation.
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