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Ingel digestion
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Chemicals Iodoethanol (Aldrich, catalog #17-685-0, stored over copper granules and should be colorless) Triethylphosphine (Aldrich, catalog #24-527-5, handled in accordance with the material safety data sheet) 1M ammonium carbonates (Sigma catalog #A-8045, pH 11, adjusted with ammonium hydroxide) Gel reagent: 50% 0.1M ammonium carbonate, 48.75% acetonitrile (by volume), 1% iodoethanol, and 0.25% triethylphosphine (final pH 10). In-gel digestion procedure 1. Coomassie blue-stained gel pieces were placed in Eppendorf centrifuge tubes. 2. The gel pieces were immersed in enough reagent to completely cover the gel pieces (usually 100μl). 3. Cap the tubes and incubate the samples at 37 °C for 60min. 4. Discard the reagent and dehydrate the samples with 100μl of pure acetonitrile for approximately 5min. 5. Dry the gel pieces under vacuum for 15min. 6. Rehydrate the gel pieces with a trypsin solution (usually 5–10μl) and incubate overnight at 37 °C. 7. Extracted peptides with a 1-M solution of O-methylisourea (pH 10) for 60 min at 37 °C to convert lysines to homoarginine and were desalted with a C-18 Ziptip. LC/MS/MS analysis of tryptic peptides Inject 10μl Tryptic digests onto a 1×50-mm C-18 reverse-phase column, and elute peptides with a gradient from 5 to 45% CH3CN developed over 120min at a flow rate of 50μl/min. Interface the column with a Deca-XP+ ion trap mass spectrometer (ThermoFinnigan), and collect data in the triple-play mode. Search MS/MS spectra against a nonredundant protein database with SEQUEST. |
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