Quantitative Proteomic Analysis of Age-Related Differences

Detailed procedures were described in our previous publications [13 (link)–15 (link)]. Briefly, 100 µg of each protein sample was prepared for trypsin digestion followed by alkylation with iodoacetamide. After chloroform–methanol precipitation, each protein pellet was digested with 1 µg trypsin overnight at 37 °C. Tryptic peptides were labeled using one of the three tandem mass tags (TMT) 6-plex reagent sets (Thermo Scientific Pierce). An equal amount of each TMT-labeled sample was combined in a single tube with SepPak purified (Waters, Ireland) using acidic reverse-phase conditions. The fractionated, labeled peptide mixtures were run on a Dionex U3000 nano-flow system (Thermo Fisher Fusion Orbitrap mass spectrometer). Chromatography was conducted in a “trap-and-load” format using an EASY-Spray source. The entire run had a flow rate of 0.3 µL/min and electrospray was achieved at 1.8 kV. The 3 runs of each age group were searched using the SEQUEST HT node of Proteome Discoverer 2.4 (Thermo Scientific). The Protein FASTA database was the Mus musculus, SwissProt tax ID = 10′090, version 2017–10-25 containing 25,097 sequences. Using a false discovery rate (FDR) of < 1%, only one unique high-scoring peptide of an identified protein was needed for addition in our results. Proteome Discoverer was also used to determine the quantitative differences between biological groups.