Protein Protocols Protein Protocols

512 Stone and Williams
3. Sequencing grade, modified trypsin (Promega): dissolve the 20-µg aliquot (obtained from
the manufacturer) in 200 µL 1 mM HCl to make a 0.1 mg/mL stock solution that appears
to be stable for at least 6 mo at –20°C.
4. Endoproteinase Glu-C: dissolve the 50-µg aliquot from the manufacturer in 500 µL
50 mM NH 4HCO 3. According to the manufacturer, the dissolved enzyme is stable for
1 mo at –20°C.
5. Lysyl endopeptidase: dissolve 2.2 mg as purchased in 2.2 mL of 2 mM Tris-HCl, pH 8.0,
to make a 1 mg/mL stock, which according to the manufacturer is stable for at least 2 yr
when stored at –20°C. More dilute solutions are made by adding 10 µL of this 1 mg/mL
stock solution to 90 µL 2 mM Tris-HCl, pH 8.0, for a 0.1 mg/mL stock.
6. Pepsin (Sigma Chemical Co., St. Louis, MO): dissolve in 5% formic acid (Baker) at a
concentration of 0.1 mg/mL.
7. Digestion buffer for in-solution digestion: 8 M urea, 0.4 M NH 4HCO 3. Prepare by dissolving
4.8 g Pierce Sequanal-Grade urea and 0.316 g Baker ammonium bicarbonate in H 2O to
make a final volume of 10 mL.
8. 50% CH 3CN/0.1 M Tris-HCl, pH 8.0: prepare by diluting 5 mL 1.0 M Tris-HCl, pH 8.0
(12.1 g Tris-HCl dissolved in 100 mL H 2O with the pH adjusted to 8.0 with HCl) with
20 mL H 2O and 25 mL 100% acetonitrile.
9. In gel digestion buffer: 0.1 M Tris-HCl, pH 8.0/0.1% Tween 20. Prepared by adding 1 mL
1.0 M Tris-HCl, pH 8.0, and 10 µL polyoxyethylene-sorbitan monolaurate (Tween 20
from Sigma Chemical Co.) to 9.0 mL H 2O.
10. Cysteine modification buffer: 0.1 M Tris-HCl, pH 8.0/60% CH 3CN is prepared by diluting
5.0 mL 1.0 M Tris-HCl, pH 8.0 with 15 mL H 2O plus 30 mL 100% CH 3CN.
11. 45 mM DTT (Pierce Chemical Co.) solution for protein reduction: dissolve 69 mg DTT in
10 mL H 2O.
12. 100 mM iodoacetic acid (IAA) (Pierce Chemical Co.) solution for alkylation: dissolve
185.9 mg IAA in 10 mL H 2O.
13. 200 mM methyl 4-nitrobenzene sulfonate (Aldrich Chemical Co.) solution for cysteine
modification: is made by dissolving 0.0434 g methyl 4-nitrobenzene sulfonate in 100%
CH 3CN. This solution is made immediately before use and is not stored.
14. 0.1% TFA/60% CH 3CN solution for peptide extraction from the gel slices: add 50 µL
100% trifluoracetic acid (TFA) to 20 mL H 2O and 30 mL 100% CH 3CN.
3. Methods
3.1. Enzymatic Digestion of Proteins
3.1.1. Digestion of Proteins in Solution
3.1.1.1. SAMPLE PREPARATION FOR IN SOLUTION DIGESTION
Proper sample preparation is critical both in avoiding sample loss and in ensuring
successful digestion (see Note 1). If the sample contains a sufficiently low level of salt
and glycerol (such that the concentration of salt and glycerol in the final digest will be
less than the equivalent of 1 M NaCl and 15% glycerol, respectively), it may simply be
reduced to dryness in a SpeedVac prior to carrying out the digest in the tube in which it
was dried. Samples containing higher levels of salts, glycerol, and/or detergents, such
as SDS, may be precipitated using either trichloroacetic acid (TCA) or acetone in order
to remove the salts, glycerol, and detergents. To TCA-precipitate the protein, 1/9th vol
of 100% TCA is added to the sample prior to incubating on ice for 30 min. The sample
is then centrifuged (10,000g/15 min) and the supernatant is removed. Residual TCA is