Team:Macquarie Australia/WetLab/Protocols/Trypsin

Manual Trypsin In-Gel Digestion

MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS

• Coommassie de-stain gels: by washing briefly with 200µL NH4HCO3 (Solution A) to make sure gel pieces are at the correct pH.
• Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each well. Vortex to mix and incubate for 10 minutes. Remove liquid.
• Repeat step 2. Gel pieces should be clear at this stage. If they are still blue, repeat as necessary until color is gone.
• Wash for 5 min with 50µL of 100% Acetonitrile (Solution C) to dehydrate gel pieces. Vortex during incubation.
• Remove Acetonitrile, then let air-dry for 10 min. The gel pieces should be noticeably shrunken and probably white.
• Reduction and Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D). Let proteins reduced for 45-60 min in 37^oC incubator.
• Remove DTT solution and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3 (Solution E). Incubate for 45 min in dark place at room temperature.
• Remove iodoacetamide, discard.
• Wash gel pieces with 200ul of NH4HCO3 (Solution A) for 5 min with vortexing. before adding 100ul of 100% Acetonitrile (Solution C).
• Remove liquid after 5 min, discard.
• Wash gel pieces with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min.
• Dehydrate with 100 ul 100% Acetonitrile (Solution C) for 5 min as above.
• Remove remaining liquid and let the gel dried.
• Trypsin Digestion: Prepare trypsin mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.
• Add 30µL (or more if required) of mixed trypsin solution to cover the gel pieces.
• Allow 30 min for gel rehydration at 4^oC (on ice).
• Digest overnight at 37^oC.
• Peptide Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml Eppendorf tubes.
• To the gel pieces, add 50 µL of 50% acetonitrile / 2% formic acid, incubate 30 min. Spin, remove supernatant and combine with initial digest solution supernatant. Please note that total volume may vary depending on the gel sizes.
• Vortex the extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.
• Spin at 14,000 rpm for 30 min to remove any micro particulates.
• Transfer the supernatant to a fresh 0.65ml eppendorf tube for storage at 4^oC fridge OR directly into PCR plate for Mass spec for analysis.

Reagents

• Buffer 100mM Ammonium Bicarbonate
  • ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill first medium size reservoir.

• Reduction: - Dithiothreitol (DTT)
  • Add 15 mL of 100 mM ammonium bicarbonate to 23.1 mg of DTT to give a solution of 10 mM DTT.

• Alkylation - Iodoacetamide(DTT)
  • Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide to give a solution of 55 mM iodoacetamide - 15 mL of each is enough for up to 96 samples.

• Peptide Extraction Solution - 2% Formic acid/ 50% Acetonitrile solution
  • Add 2000µL of formic acid and 50 mL of acetonitrile to 75 mL of ddH2O. 15 mL is enough for up to 96 samples.

• Dehydration-Acetonitrile
  • A full square reservoir is plenty for up to 96 samples with dehydration and/or Coomassie destaining.

• Trypsin
  • Trypsin is prepared by adding 200µL of Resuspension Buffer into one vial containing 20ug of Proteomics grade trypsin.
  • Add 1.2ml of 50mM NH4HCO3 into each trypsin vial (final concentration of Trypsin is 16ng/µL). Mix thoroughly.
  • This will provide enough Trypsin for 46 digestions.