In-Gel Protein Digestion

The procedure was developped at the UCSF mass spectrometry Facility, it is reported here for your convenience.

Sample Preparation

Wearing gloves and sleeve protectors, wipe down ALL surfaces in the hood with methanol/water moistened lint-free cloth, including the outside of all your tubes (make sure to not wipe off the labeling!), the outside and inside of the speed-vac, centrifuge, tube racks, bottles etc. Wipe razor blades with methanol-soaked lint-free cloth.

  1. Prepare the following solutions:
    • 25 mM NH4HCO3 (100 mg/50 ml), pH 7.5
    • 25 mM NH4HCO3 in 50% CH3CN.
    • 50% CH3CN, 5% formic acid (may substitute TFA or acetic acid).
    • 12.5 µg/mL (ng/mL for low concentration of peptides) trypsin in 25mM NH4HCO3 (freshly diluted).
  2. Dice each gel slice into small pieces (1 mm2) and place into 0.65 mL siliconized tubes (PGC Scientific).
  3. Add ~100µL (or enough to cover) of 25mM NH4HCO3/50% CH3CN and vortex for 10 min.
  4. Discard supernatant.
  5. Repeat steps 3 and 4 two times. For Coomassie blue stained gels, if gel pieces are still very blue after 1st washing, you can rehydrate the gel pieces with ammonium bicarbonate before repeating the washes.
  6. Speed Vac the gel pieces to complete dryness (~ 20 min).

Reduction and Carboxymethylation

  1. Prepare fresh solutions:
    • 10 mM DTT in 25 mM NH4HCO3 (1.5 mg/mL)
    • 5 mM iodoacetamide in 25 mM NH4HCO3 (10 mg/mL)
  2. Add 25 mL (or enough to cover) 10 mM DTT in 25 mM NH4HCO3 to dried gels. Vortex and spin briefly. Allow reaction to proceed at 56 °C for 1 hr.
  3. Remove supernatant, add 25 mL 55 mM iodoacetamide to the gel pieces. Vortex and spin briefly. Allow reaction to proceed in the dark for 45 min. at room temperature.
  4. Remove supernatant (discard). Wash gels with ~100 mL NH4HCO3, vortex 10 min, spin.
  5. Remove supernatant (discard). Dehydrate gels with 25 mM NH4HCO3 in 50% ACN, vortex 5 min, spin. Repeat one time.
  6. Speed Vac the gel pieces to complete dryness (~20 min). Proceed with Trypsin digest.
    For low-level proteins (<1 pmol), especially those separated by 1D SDS-PAGE, reduction and alkylation is recommended. These procedures are performed after step 6.
    1. Prepare fresh solutions:
      • 10 mM DTT in 25 mM NH4HCO3 (1.5 mg/mL)
      • 5 mM iodoacetamide in 25 mM NH4HCO3 (10 mg/mL)
    2. Add 25 mL (or enough to cover) 10 mM DTT in 25 mM NH4HCO3 to dried gels. Vortex and spin briefly. Allow reaction to proceed at 56 °C for 1 hr.
    3. Remove supernatant; add 25 mL 55 mM iodoacetamide to the gel pieces. Vortex and spin briefly. Allow reaction to proceed in the dark for 45 min. at room temperature.
    4. Remove supernatant (discard). Wash gels with ~100 mL NH4HCO3, vortex 10 min, spin.
    5. Remove supernatant (discard). Dehydrate gels with 25 mM NH4HCO3 in 50% ACN, vortex 5 min, spin. Repeat one time.
    6. Speed Vac the gel pieces to complete dryness (~20 min). Proceed with trypsin digest.

In-Gel Digest Procedure

Estimate dried gel volume (V) in mL. Add (3 x V) mL trypsin solution. This volume will vary from sample to sample, but on average ~25-60 µL is sufficient. Vortex for 10 min., then sonicate for 3 min.

  1. Incubate at 4 °C for 30 min. Add 25 mM NH4HCO3 as needed to cover the gel pieces. If trypsin solution remains in the sample, pipet it off and replace with 25 mM NH4HCO3.
  2. Spin briefly, cover tubes with parafilm and incubate at 37C overnight (16-20 hrs).
  3. Extraction of Peptides
  4. Briefly vortex and spin the digest. Add 25-100 mL H2O, vortex 10 min, spin, sonicate for 5 min. (Alternatively, for small samples, add 5 mL H2O, vortex and sonicate).
  5. FOR MALDI: Remove 2 mL and mix with 2 mL 30% CH3CN /5% formic acid. Use 0.5 mL of the peptide mixture plus 0.5 mL DHB for preliminary analysis.
  6. Continue extraction with remaining sample.
  7. Transfer the digest solution (aqueous extraction) into a clean 0.65 mL siliconized tube to which 5 mL 50% CH3CN, 5% formic acid has been added.
  8. To the gel pieces, add 50 µL (enough to cover) of 50% CH3CN /5% formic acid, vortex 10 min., spin, sonicate 5 min. (only sonicate for the first organic extraction). Pool extracted peptides together in one tube. Repeat two times.
  9. Vortex the extracted digests, spin and Speed Vac to reduce volume to 10 µL.
  10. Add 100 µL H2O, vortex, and Speed Vac to ~10 µL. Add 2-5 µL 50% CAN, 5% formic acid.
  11. Utilize 1µL of the unseparated digests for analysis by MALDI.

    Preliminary MALDI results will determine whether or not a cleanup using C18 ZipTips  (Millipore) or HPLC separation of the peptides is required.

Matrices for unseparated digests:
a-cyano-4-hydroxycinammic acid in 50% CH3CN 1% TFA (10 mg/mL).
2,5-dihydroxybenzoic acid (DHB), in 20% CH3CN /1% TFA (10 mg/mL).

 

References:

Rosenfeld, et al., Anal. Biochem. (1992) 203, 173-179.

Hellman, et al., Anal. Biochem. (1995) 224, 451-455.

Prcedure developped at the UCSF Mass Spectrometry facility

Last modified: Mar 26, 2013
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