Following SDS-PAGE, gel lanes were sliced and subjected to in-gel digestion with trypsin58 (link) with modifications11 (link). Peptide samples were analysed by liquid chromatography (LC)-tandem MS using a nanoACQUITY UltraPerformance LC system (Waters) coupled online to an LTQ Velos mass spectrometer (Thermo Fisher Scientific) or using an UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific) coupled online to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were concentrated and desalted on a Symmetry C18 preparative column (20 mm × 180 μm, 5-μm particle size; Waters) and separated on a bridged ethyl hybrid C18 analytical column (250 mm × 75 μm, 1.7-μm particle size; Waters) using a 45-min linear gradient from 1% to 25% or 8% to 33% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nl/min. Peptides were selected for fragmentation automatically by data-dependent analysis.
Protocol detail
Proteomic Analysis via SDS-PAGE and LC-MS/MS
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Other organizations : Wellcome Centre for Cell-Matrix Research, University of Manchester, Edinburgh Cancer Research
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Variable analysis
independent variables
- Trypsin digestion with modifications
- Peptide samples analyzed by liquid chromatography (LC)-tandem MS
- Symmetry C18 preparative column (20 mm × 180 μm, 5-μm particle size; Waters)
- Bridged ethyl hybrid C18 analytical column (250 mm × 75 μm, 1.7-μm particle size; Waters)
- 45-min linear gradient from 1% to 25% or 8% to 33% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nl/min
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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