LC-MS/MS identification and quantitative analysis of protein.

For preparation, 3-mm-long muscle fibers were dissected and their CSAs calculated as above. These fibers were then placed in tubes containing 25 μl Tris-Triton lysis buffer (10mM Tris [pH 7.4], 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate). Prior to enzymatic digestion and labeling, the samples were loaded into a stack gel for lysis buffer cleanup to eliminate chemical interference at the labeling stage and to compress the whole proteome into a single band. Sample volumes were dried by half in a SpeedVac (Thermo Fisher Scientific) with the volume replaced by Laemmli buffer (2×) and boiled for 10 minutes at 96°C. Reduced samples were loaded onto a 10% Bis-Tris NuPAGE gel and resolved for 10 minutes (100 V; 59 mA; 6 W) to “stack” the whole sample into a single band. Protein bands were visualized using Imperial protein stain (Thermo Fisher Scientific).

For digestion and peptide labeling with tandem mass tag (TMT), in-gel reduction, alkylation, and digestion with trypsin were performed on all the samples prior to subsequent isobaric mass tag labeling (43). Each sample was treated individually with labels added at a 1:1 ratio.

For LC-MS/MS tandem mass spectrometry, the combined TMT-labeled peptide samples were resuspended in a solution containing water:acetonitrile:trifluoroacetic acid (98%:2%:0.05%) and analyzed by LC-MS/MS. Chromatographic separations were performed using an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). A 10-μl injection of peptides was resolved by reversed phase chromatography on a 75-μm C18 column (50 cm) using a 3-step linear gradient of acetonitrile in 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 250 nl/min over 120 minutes. The eluate was ionized by electrospray ionization using an Orbitrap Fusion Lumos (Thermo Fisher Scientific) operating under Xcalibur v4.1. The instrument was programmed to acquire in automated data-dependent switching mode. The selection of precursor ions was based on their intensity for sequencing by higher-energy C-trap dissociation (HCD) for peptide identification and reporter ion fragmentation. Selection of precursor ions was also based on their intensity for sequencing by HCD in a TopN method. The MS/MS analyses were conducted using higher than normal collision energy profiles that were chosen based on the mass-to-charge ratio (m/z) and the charge state of the peptide. To increase fragmented peptide coverage and reporter ion intensities, a further synchronous precursor scan (SPS) of the top 5 most intense peaks using MS3 (Thermo Fisher Scientific) was performed.