ShotgunProteomicsWithTFE protocol

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Protocol: Lysis

  1. Resuspend cell pellet in 500 μL ~ 2 ml lysis buffer of choice based on sample requirements.
    • Buffer compatibility is largely based on digestion, chromatography, and mass spec. concerns, and should be verified before using a buffer for the first time.
    • In general, try to maintain a ~ neutral pH, minimize salt concentration, and avoid detergents and anything else that might stick to the proteins and peptides.
    • As an example, HeLa cell lysis buffer consisted of 50mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA, and Protease Inhibitor Cocktail Set 1 (Calbiochem).
  2. Lyse cells in 2 ml dounce homogenizer (~25 strokes), then centrifuge sample at max speed for 5 min. at 4 °C.
  3. Transfer the supernatant, which contains the soluble (cytosolic) fraction, to a new tube.
  4. If interested in the insoluble (membrane-bound, nuclear, etc.) fraction, wash it in PBS or lysis buffer and centrifuge again to pellet and save (it is not clear yet whether this TFE protocol is compatible with the insoluble fraction, it may be necessary to use a mass spec compatible detergent such as Rapigest). Even if the insoluble fraction is not of interest, it is always a good idea to hang onto this fraction until you are sure that lysis was successful, as unbroken cells will be found in the pellet. Lysate and insoluble fractions should be stored at -80 °C.
  5. Measure the protein concentration. For digestion, you will need 50 μL at 1 ~ 4 mg/ml (I prefer to start at 2 mg/ml). It is a good idea to aliquot the lysate into fractions based on the concentration so that the samples do not undergo repeated freeze/thaw.

Protocol: Digestion

  1. Add 50 μL trifluoroethanol (TFE) to 50 μL sample and vortex to get final concentration of 50% TFE.
    • TFE is stored in a flammable solvent cabinet.
  2. Add 11 μL of 150 mM DTT to get 15 mM DTT in total.
    • DTT is an unstable reducing agent. It is best to make stock solution fresh, or use within 1-2 weeks (stored at -20˚C).
    • Measure 0.006 g of DTT, then add 300 μL of water.
  3. Heat to 55 ˚C for 45 min.
  4. Cool samples to room temperature (~5 min).
  5. Add 12 μL of 550 mM IAM to get 55 mM IAM in total.
  6. Incubate in the dark at room temp for 30 min.
    • Leave it inside the drawer under the bench.
  7. Dilute sample by adding 880 μL of 50mM Tris + 2 mM CaCl2, pH 8.0 to get 5% TFE.
  8. Add trypsin to a final concentration of 1:25-1:50 enzyme:protein (2 µg trypsin for 1-2 mg/ml).
    • Resuspend 20 µg trypsin (Sigma) in 200µl of 1mM HCl.
    • Aliquot into 20 μL stock solutions (2 µg of trypsin), can be stored for several months at -80˚C.
  9. Incubate at 37 ˚C for 4 ~ 5 hrs.
  10. Add 10 ˚C of formic acid (to get 1% vol./vol. in total) to stop digestion.
    • Formic acid is stored in a flammable solvent cabinet.
    • At this point, samples can be stored overnight in -80 ˚C freezer.
  11. Dry sample to 10-20 μL with SpeedVac (remove the red cap before running SpeedVac). It takes about 4 ~ 5 hrs.
  12. Resuspend to 150 μL in Buffer C (95% H2O + 5% Acetonitrile + 0.1% formic acid).
  13. Clean up samples on C18 tips (Thermo) according to manual.
    1. Wash the resin 3 times, with 50 μL of 60% Acetonitrile solution (60% of Buffer B + 40% of Buffer A).
    2. Wash the resin 3 times, with 50 μL of Buffer A.
    3. Load the sample. 90 {μL max. at a time.
    4. Wash the sample 3 times, with 50 μL of Buffer A.
    5. Elute the sample with 50 μL of 60% Acetonitrile solution. Repeat elution step twice to get 100 μL as the final solution.
  14. Dry out sample to about 10-20 μL with SpeedVac (20-30 min).
  15. Resuspend with 100 μL Buffer C (about 120 μL in total).
  16. Load sample onto Amicon Ultra-0.5 (10 kDa) to get rid of junks
    • Before loading a sample, wash the column with 500 uL of Buffer C (twice).
    • After washing, get rid of residual Buffer C (10-20 uL) inside the filter by pipette.
  17. Centrifuge at 14,000 xg (rcf) for 12 min at 4 deg C. Take the eluate.
  18. The sample is now ready for LC/MS analysis. After finishing all steps, transfer 50 μL of filtered sample onto vial insert.
    • National Scientific Target DB Vial C4000-1W with a cap(C4000-54B)
    • Agilent Vial Insert (5181-1270)

Comments

  • Following lysis, sample prep is the same for all samples.
  • Maintain all samples on ice throughout lysis.

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