Martin Humphries
LAB

Protocols - 96-well plate in-gel digestion method for silver and coomassie stained proteins


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  • Perform as much as possible in the MS fume cupboard
  • Make solutions fresh from stocks for each digestion

Solutions


  • 1M NH4HCO3: 7.9g in 100ml Milli-Q H20
  • 25mM NH4HCO3: 1ml of stock 1M NH4HCO3 in 40mls Milli-Q H20
  • 50% (v/v) Acetonitrile / 50% (v/v) 25mM NH4HCO3: 5mls of each stock
  • DTT (Sigma D5545): 1.54mg DTT/1ml 25mM NH4HCO3
  • Iodoacetamide (Sigma I149): 8mg/1ml 25mMNH4HCO3
  • -100x Trypsin Stock (125ng/µl) (Promega V511A): 160µl trypsin buffer plus 20μg trypsin
  • 1x Trypsin (1.25ng/µl): 50µl trypsin stock in 5ml 25mM NH4HCO3.
  • 20mM NH4HCO3: 0.5ml of stock 1M NH4HCO3 in 25mls Milli-Q H20
  • 50% (v/v) Acetonitrile / 5% (v/v) Formic acid: 5mls acetonitrile plus 0.5ml formic acid plus 4.5ml Milli-Q H20
  • O.1% (v/v) Formic Acid: 100ul formic acid in 100mls Milli-Q H20


Day 1


Discard everything in the collection plate. Perform as much as possible in the MS fume cupboard.

  1. Assemble the plate with the perforated plate on the top and the storage plate underneath and label the plate.
  2. Switch on vacuum. Switch on both boxes on the floor first. This needs to get to -118°C.
  3. Excise the bands of interest from the gel using a clean razor, place gel pieces into a well in the perforated plate.
  4. Make fresh 25mM NH4HCO3 (50ml) from stocks.
  5. Spin plate at 1500rpm (2min) using the 96 well plate rotor to remove the water.

  6. The liquid is centrifuged into the storage wells which are then emptied. Check for wells that do not empty.
  7. Perform two 5min washes with 50ul 50% acetonitrile and 50% 25mM NH4HCO3.

  8. Acetonitrile is organic so use special waste container. At that stage the gel must be totally transparent, if any blue is left do a 3rd wash.
  9. Wash 2 time with 50µl of acetonitrile for 5min, centrifuge.
  10. Dry the pieces in a vacuum centrifuge for 30min in the 96well plate rotor.
  11. Prepare DTT solution and iodoacetamide solution.
  12. Add 50µl of DDT solution to gel and reduce proteins for 1 hour at 56°C. Centrifuge.
  13. Wash with 50µl 25mM NH4HCO3 for 10min, centrifuge.
  14. Wash with 50µl acetonitrile for 5min, centrifuge
  15. Wash with 50µl 25mM NH4HCO3 for 5min, centrifuge
  16. Wash again with 50µl acetonitrile, centrifuge out the liquid phase and completely dry the gel pieces for 25min in the vacuum centrifuge.
  17. Replace the collection plate by a fresh one.
  18. Add 5µl of 10x trypsin to each well then incubate for 40mins at 4°C
  19. Add 45µl of 25mM NH4HCO3
  20. Place well plate in a container incubate overnight at 37°C.
  21. Discard used solutions, switch off the pump.


Day 2


Keep everything in the collection plate.

  1. Centrifuge the plate.
  2. Extract proteins with 50µl 100% Acetonitrile 0.2% formic acid for 30min at RT, centrifuge.
  3. Extract proteins with 50µl 50% Acetonitrile 0.1% formic acid for 30min at RT, centrifuge.
  4. Evaporate completely pooled supernatants with the vacuum centrifuge.
  5. Label ms-ms tubes.
  6. Re-suspend peptides into 20µl of 5% Acetonitrile 0.1% formic acid.
  7. Store peptides bellow-20°C.


Avoiding PEG contamination


PEG in your samples severely affects the quality of data that the MS can achieve and causes no end of problems for the facility trying to eradicate it from the machines. The contamination can come from many sources, the risk of which can be reduced with the following:



  1. Make fresh solutions for trypsin digestion from stocks every time and only making what you require
  2. Be careful weighing out substances like DTT and Iodoacetamide. Weigh into a clean eppendorf from a bag that is only for MS use. These are usually kept in the MS drawer by the hood. If in doubt open a new bag. Also make sure the balance is clean before using it and try not to use a balance known to be used by people using PEG
  3. Keep the MS hood clean and free of clutter. Only have the tips etc that you need at any one time and remove the boxes and clean the hood after use
  4. Be careful running gels and preparing samples before digestion. Use a clean square petri dish or tube and minimise the time it is open to air
  5. All solutions left in the hood that are not the stock bottles should be disposed off after use. Do not reuse old falcon tubes as substances can leach out into your solution over time

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