Tmtpro 16plex label

Quadriceps muscle (n = 3 biological replicates for each treatment group) from 35 day old mice were lysed in RIPA buffer with inhibitors and 0.5% SDS. Proteomics analysis was conducted by the Network Biology Collaborative Centre (NBCC) at Lunenfeld-Tanenbaum Research Institute (Toronto, ON). A total of 50 μg of protein processed using S-traps micro spin columns (ProtiFi) per manufacturer’s protocols and digested with 2 μg of trypsin for 1 h at 47 °C. Tandem mass tag (TMT) labelling was performed to quantify all samples. Approximately 10 μg of each sample was labelled with 80 μg of its respective TMTpro 16plex label (ThermoFisher) per manufacturer protocols. Samples were lyophilized and 1/20th of each labelled sample was combined. High pH fractionation was performed and 3/4 of each labelled sample was combined for ~ 96 μg and fractionated with Pierce High pH fractionation kit (Cat#: 84868) per kit instructions. Trapped and trypsin-digested peptides were acquired on a 90 min gradient using an Orbitrap Fusion Lumos Tribrid mass spectrometer. Data was searched with SequestHT using the UP000000589 proteome (Mus musculus) generated by Uniprot, post-processed with Percolator, and analyzed using Proteome Discoverer 2.2 (Thermo Scientific). Metascape was used for GO term enrichment analysis of differentially expressed proteins (log2FC > 0.585, p value < 0.05).