Heart and skeletal muscle of young adult and early aging mice

Left cardiac ventricles and quadriceps femoris muscles were collected from young adult (16-17 wk) and early aging (86 wk) C57BL6/J mice (n=4, 2 male 2 female). Proteins were extracted in 200 µL RIPA buffer and Halt protease/phosphatase inhibitor with a hand-held sonicator followed by centrifugation, reduced with dithiothreitol, alkylated with iodoacetamide, then digested with modified sequencing grade trypsin overnight at 37degC using a filter-assisted sample preparation protocol. The peptides were fractionated using Thermo high-pH reversed phase columns into 8 fractions. Label-free bottom-up mass spectrometry was performed using data dependent acquisition on a Thermo Orbitrap Q-Exactive HF instrument using typical settings.