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

WT C57BL6 mice (N=3 per group) carried out a 90 min treadmill running protocol, starting with a speed of 4 meters/min, with 2 meters/min increments every 10 min until 24 meters/min (Exercise). 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. Raw data were searched using Mascot (Matrix Science, London) through the Proteome Discoverer software against the Mus Musculus Swissprot database. Search conditions included dynamic modifications for methionine (Oxidation) and arginine (Label:13C(6)) and indicated quantification using SILAC (Lys6). Searches were performed using either trypsin or LysC as the enzyme and mean H/L calculated for each protein and expressed as a ratio of ratios Exercise/Rest.