Tandem Mass Tag Labeling and Fractionation

The 6-plex tandem mass tag (TMT) labeling was carried out according to Thermo Scientific’s TMT Mass Tagging Kits protocol. Each of the TMT 6-plex label reagents was reconstituted in 45 µL of acetonitrile (ACN), and the digested peptides from each sample were incubated with a specific tag (tags 128N, 129N, 129C used for 3 MG.MSTN^+/− samples and tags 130N, 130C, 131 used for 3 MG.WT samples, respectively) for 1 h in room temperature (Figure 12). And then the samples were pooled and dry-out to cation exchange chromatography. The hpRP chromatography was carried out as described previously (Yang et al., 2011 (link)). Forty-eight fractions were obtained at 1 min intervals and pooled into 6 fractions based on UV absorbance at 214 nm and with a multiple fraction concatenation strategy (Wang et al., 2011 (link)). The trypsin peptides labeled in each component are used for global proteomic analysis of nanoLC-MS/MS analysis.