Effect of treadmill exercise on the mouse gastrocnemius proteome assessed via spike-in SILAC quantitative proteomics

Resting controls were placed on the motionless treadmill for the same duration. After euthenasia, the gastrocnemius muscle was homogenised in lysis buffer (50mM TRIS (pH 7.5), 1mM EDTA, 150mM NaCl, 0.1% Sodium deoxycholate, 1% NP-40, 1x protease inhibitor cocktail (Sigma) and combined at a 1:1 ratio with a mixed muscle homogenate processed from fully labelled C13Lys6 SILAC mice.

Lysates were fractionated via 1D SDS PAGE and cut into 10 sections, washed and rehydrated in 100mM ammonium bicarbonate before reduction and alkylation in 10ul DTT and Iodoacetamide respectively. Digestion was carried out in either LysC or Trypsin with the resultant peptides dried and resuspended in 2% acetonitrile, 0.1% formic acid. Data from samples were then acquired via LC/MS-MS on an Orbitrap Elite (Thermo Scientific) using a top 15 method with CID fragmentation.