Contributed by Lise R. Riviere and Paul Tempst
Current Protocols in Protein Science (1995) 11.1.1-11.1.19
Protein is solubilized in an appropriate digestion buffer and subjected to enzymatic
proteolysis. Fragmentation is monitored by SDS-PAGE or RP-HPLC, followed by
preparative RP-HPLC of the completed digest. The procedure is recommended for
checking protease sensitivity of unknown proteins or for preparing proteolytic fragments
that can be sequenced when working with proteins already shown to be sensitive to a
particular protease. The minimal amount of protein required is 100 pmol.
Materials
100 pmol to 5 nmol protein sample, as pellet or solution
1 × and 10× digestion buffer (see Table 11.1.1)
20% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS),
20% octyl glucoside, or 20% Nonidet P-40 (NP-40; Calbiochem)
100% acetonitrile
1 μg/μl enzyme stock (see Support Protocol 1; store at −20°C)
Trifluoroacetic acid (TFA; Pierce)
Solvent A: 0.1% (v/v) TFA in water
Solvent B: 0.09% (v/v) TFA in 70% (v/v) acetonitrile (Burdick & Jackson)
Sonicator bath (Branson)
Phast Gel system (Pharmacia), optional
HPLC system, with C18 or C4 reversed-phase column, 4.6-mm or 2.1-mm (e.g.,
Vydac);UV detector; chart recorder
Additional reagents and materials for SDS-PAGE (UNIT 10.1), gel staining
(UNIT 10.5), and HPLC analysis of peptides (UNIT 11.6)
1. Dissolve protein pellet in minimal volume of appropriate 1× digestion buffer. If
protein sample is already in solution, add 0.1 vol of 10× digestion buffer.
Final protein concentration must be >1 μg/20 μl. If sample is too dilute and volume is over
0.5 ml, use Centricon microconcentrator (see manufacturer’s instructions) to reduce the
volume.
2. Incubate sample 5 min at 37°C, then sonicate 1 min. Repeat until pellet is completely
dissolved. If pellet does not dissolve, add CHAPS (2% final), octyl glucoside (2%
final), NP-40 (2% final), or acetonitrile (20% to 40% final; Table 11.1.1) to digestion
solution.
These solubilization agents do not interfere with digestion (see Table 11.1.1).
NOTE: Digestion solution should not contain protease inhibitors. All proteases are
inhibited by leupeptin. In addition, trypsin, chymotrypsin, endoproteinase Lys-C, endoproteinase
Glu-C, subtilisin, and elastase are inhibited by phenylmethylsulfonyl fluoride
(PMSF), diisopropyl fluorophosphate (DFP), and aprotinin; thermolysin and endoproteinase
Asp-N are inhibited by EDTA.
3. Transfer 1 μl sample to pH indicator strip. If pH is not correct, add sufficient 10×
digestion buffer to increase buffer strength by 100 mM (see Table 11.1.1) and recheck
the pH.
For pepsin, the pH must be 2.0. Subtilisin and endoproteinase Lys-C work well in a pH
range of 7 to 10.5. For all other proteases, pH should be 8.0 to 8.5.
4. Add 1 μg/μl enzyme stock solution to a final enzyme/substrate ratio of 1:50 to 1:10
(w/w).
Final enzyme concentration should be >1 μg/100 μl.
5. Incubate at 37°C for the amount of time recommended for the selected protease (see
Table 11.1.1).
6. Determine extent of digestion by analyzing 0.5 to 1 μg digest by SDS-PAGE (e.g.,
using a Phast Gel). Stain with Coomassie brilliant blue (UNIT 10.5). Alternatively,
analyze 25 to 500 pmol by RP-HPLC (use a 2.1-mm column for 25 to 200 pmol
protein or a 4.6-mm column for 200 to 500 pmol protein). Keep remaining sample
on ice.
The procedure should not consume >10% of the sample. Phast Gels are useful when protein
amounts are limited.
7. If sample is digested, proceed to preparative RP-HPLC (step 8). If sample is not
digested, check pH and adjust if necessary. Add a second aliquot of enzyme and
incubate at 37°C for an additional 30 min to full incubation time. Check extent of
digestion as in step 6.
If sample is still not digested after incubation with a second aliquot of enzyme, try a different
enzyme. If the second enzyme is ineffective, denature the substrate (see Alternate Protocol
1 and Alternate Protocol 2).
If HPLC analysis cannot be performed immediately, TFA should be added to a final
concentration of 2% and the sample should be stored at −70°C. Digestion samples may be
kept indefinitely under these conditions.
8. Using a clean Hamilton syringe, inject remaining digested sample onto column.
Program HPLC to run isocratically at 5% solvent B until baseline returns to its
original position. Set flow rate to 100 μl/min for a 2.1-mm column or 1 ml/min for
a 4.6-mm column. Run gradient as follows:
5% to 50% solvent B in 45 min;
50% to 100% solvent B in 25 min.
A 4.6-mm column can easily accommodate 1 to 2 ml sample volume. If the sample contains
organic solvent, it should be diluted five-fold with solvent A.
See UNIT 11.6 for further details on HPLC separation of peptides.
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