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Sample Preparation

Table Of Contents

  1. In-Gel Digest Procedure
  2. Methods for Trypsin Digest
  3. Methods for analysis of Proteins
  4. Methods for analysis of Peptides
  5. Alternative plate spotting method for silver stained gel spots

This is the current procedure for In-Gel digests used at UAB.
(Modified from the procedure at UCSF)

In-Gel Digest Procedure

  1. Prepare the following solutions:
    25 mM NH4HCO3 (100 mg/50 ml)
    25 mM NH4HCO3 in 50% ACN
    50% ACN/5% formic acid (may substitute TFA or acetic acid)
    12.5 ng/µL trypsin in 25mM NH4HCO3 (stored in 200 µl aliquots at -80oC in a freezer)
  2. Dice each gel slice into small pieces (1 mm2, or crush with razor blade) and place into 0.65 mL tubes.
  3. Add ~50 µL (or enough to cover) of 25 mM NH4HCO3/50% ACN, vortex and mix on the Nutator for 30 min.
  4. Extract the supernatant and transfer to a separate tube (to be discarded).
  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).
  7. Add 20 µL trypsin solution. This volume will vary from sample to sample.
  8. Incubate at room temp. for 30 min. Add 10-20 µl 25 mM NH4HCO3 if needed to cover the gel pieces.
  9. Spin briefly, and incubate at 37oC overnight (16-20 hrs).

Extraction of Peptides

  1. Briefly vortex and spin the digest add 50 µL (enough to cover) of 50% ACN/5% formic acid, vortex and mix on the Nutator for 30 min. Use 1 µL of the peptide mixture plus 1 mL CHCA for preliminary analysis. Continue extraction with remaining sample.
  2. Transfer the supernatant to a clean tube. Add 50 µL (enough to cover) of 50% ACN/5% formic acid, vortex and mix on the Nutator for 30 min. Pool extracted peptides together in one tube. Repeat one more time.
  3. Vortex the extracted digests, spin and Speed Vac to reduce volume to dryness.
  4. Add 5-10 µL 50% ACN/5% formic acid.
  5. Mix with 9µL CHCA and spot on the plate. If the protein concentration is very low, use either a 1/5 or 1/1 dilution. (An alternative spotting method using nitrocellulose can also be used - see attached

Matrices for unseparated digests:

  • a-cyano-4-hydroxycinammic acid in 50% ACN/1% TFA (10 mg/mL).
  • 2,5-dihydroxybenzoic acid (DHB), in 20% ACN/1% TFA (10 mg/mL).

References:

  • Rosenfeld, et al., Anal. Biochem. (1992) 203, 173-179.
  • Hellman, et al., Anal. Biochem. (1995) 224, 451-455.

Methods for Trypson Digest:

The protein was identified by analysis of the tryptic fragments and comparison with the NCBI database. The procedure used was adapted from that developed at UCSF (http://donatello.ucsf.edu/ingel.html). Briefly, the band was excised from a polyacrylamide gel, digested overnight with trypsin, and the peptides were extracted with 50% acetonitrile/5% formic acid. The extracts were concentrated in a speed vac and redissolved in 10 µl 50% acetonitrile/5% formic acid. The extracted peptides were mixed with the matrix a-cyano-4-hydroxycinammic acid (Aldrich, 47,687-0), spotted onto a MALDI plate, and analyzed with a Voyager DePro mass spectrometer (Applied Biosystems). The peptide masses were entered into Mascot (http://www.matrixscience.com/) and the NCBI database was searched to identify the protein.

Methods for analysis of Proteins:

Samples were analyzed in the positive mode on a Voyager Elite mass spectrometer with delayed extraction technology (PerSeptive Biosystems, Framingham MA). The acceleration voltage was set at 25kV and 10-50 laser shots were summed. Sinapinic acid (Aldrich, D13,460-0) dissolved in acetonitrile : 0.1% TFA (1:1) was the matrix used. The mass spectrometer was calibrated with apomyoglobin. Samples were diluted 1:10 with matrix, and 1 ul was pipetted onto a smooth plate.

Methods for analysis of Peptides:

Samples were analyzed in the positive mode on a Voyager Elite mass spectrometer with delayed extraction technology (PerSeptive Biosystems, Framingham MA). The acceleration voltage was set at 20kV and 100 laser shots were summed. a-cyano-4-hydroxycinnamic acid (CHCA) (Aldrich, 47,687-0) dissolved in acetonitrile : 0.1% TFA (1:1) was the matrix used. The mass spectrometer was calibrated with bradykinin, angiotensin, and neurotensin. Samples were diluted 1:10 with matrix, and 1 ul was pipetted onto a smooth plate.

Alternative plate spotting method for silver stained gel spots

MALDI plates are prepared by a method adapted from Miliotis et al. Briefly, the stainless steel plates are pre-coated with an acetone solution containing 10 mg/ml CHCA and 1 mg/ml nitrocellulose. The peptide digest sample is dissolved in 10 µl of acetonitrile / 5% formic acid (50:50) and 0.5 µl is spotted onto the matrix. The sample is washed two times with 3-5 µl 0.1% TFA and analyzed of a Voyager DePRO mass spectrometer.

Reference:

  • Journal of Neuroscience Methods.
  • Development of silicon microstructures and thin film MALDI target plates for automated proteomics sample identifications.
  • Tasso Miliotis, Gyorgy Marko-Varga, Johan Nilsson and Thomas Laurell.
  • Dept of Analytical Chemistry, Lund University, Bos 124, SE-221 00 Lund Sweden.