Muscle lysate protein (80 µg total protein) was separated on 4–20% TGX Criterion gradient SDS polyacrylamide gels (Bio-Rad, Berkeley, CA) and processed for mass spectrometry, as previously described (12 (link)). Mass spectrometry (liquid chromatography–electrospray ionization–tandem mass spectrometry [LC-ESI-MS/MS]) was performed on a Thermo Electron (San Jose, CA) Orbitrap Elite Velos Pro fitted with an EASY source (Thermo Electron, San Jose, CA). NanoLC was performed using a DIONEX/Thermo NCS-3500RS UltiMate 3000 with an EASY Spray column (Thermo Electron, San Jose, CA; 50 cm × 75 μm inner diameter, packed with PepMap RSLC C18 material, 2 μm). A “top 15” data-dependent MS/MS analysis was performed (acquisition of a full-scan spectrum followed by collision-induced dissociation mass spectra of the 15 most abundant ions in the survey scan).
Protocol detail
Muscle Proteome Analysis by LC-MS/MS
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TGX Criterion gradient SDS polyacrylamide gels
Orbitrap Elite Velos Pro
EASY source
NCS-3500RS UltiMate 3000
EASY Spray column
PepMap RSLC C18 material
Electron
Electrospray ionization mass spectrometry
Ions
Liquid chromatography
Mass spectra
Ms ms
Muscle protein
Polyacrylamide gels
Protein
Scan
Corresponding Organization : University of Arizona
Other organizations : Mayo Clinic Hospital
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Variable analysis
independent variables
- Muscle lysate protein (80 µg total protein)
- Mass spectrometry (liquid chromatography–electrospray ionization–tandem mass spectrometry [LC-ESI-MS/MS]) performed on a Thermo Electron (San Jose, CA) Orbitrap Elite Velos Pro fitted with an EASY source (Thermo Electron, San Jose, CA)
- 4–20% TGX Criterion gradient SDS polyacrylamide gels (Bio-Rad, Berkeley, CA) used for protein separation
- DIONEX/Thermo NCS-3500RS UltiMate 3000 with an EASY Spray column (Thermo Electron, San Jose, CA; 50 cm × 75 μm inner diameter, packed with PepMap RSLC C18 material, 2 μm) used for nanoLC
- No positive or negative controls were explicitly mentioned in the provided information.
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