In-Solution Tryptic Digest Protocol
Adapted from: Current Protocols in Protein Science, (1996-current) John Wiley and Sons, Inc. Brooklyn, Section 11.1.
Notes:
- Read the entire set of instructions from Promega for performing a tryptic digest. The text can be found at http://www.promega.com/tbs/tb512/tb512.html.
- Start with the simplest digest protocol (e.g., no protein denaturing or reduction) unless it is know that more stringent conditions must be used. Modify this protocol as necessary. Different proteins may have different optimal digest conditions. Be aware of the ?Stability? and ?Reaction Conditions? which are described in the text by Promega.
For a digestion, it is recommended to use tubes that have screw-on caps with an o-ring.
- Set heating block (or equivalent) to 37 ºC.
- Prepare buffer if necessary: 50 mM AMBIC (ammonium bicarbonate, NH4HCO3) buffer, pH 8.5 (or, pH between 7.5 and 9.0 is acceptable). Tris buffer is also used successfully. (It might be useful to have a 0.1 M stock of buffer pH 8.5 in the event that pH of an unknown sample has to be adjusted.) Buffer containing 1 mM CaCl2 is recommended, but not absolutely necessary, for tryptic digests.
- Determine a suitable S/E ratio
(S = substrate, i. e. , protein to be digested, E = proteolytic enzyme)
Initial recommendation for S/E is in the range of 100 to 20 (w/w). The optimal ratio must be determined experimentally. Chose 2 ratios for the first experiment and check results. Modify if necessary. The weight equivalent of the substrate refers to the total amount of protein in the sample. For example, if the concentration of the substrate is 0.15 mg/ml (0.15 ug/ul) and the amount of sample chosen for the digest is 100 ul, then the total amount of substrate is 0.15 ul/ul x 100 ul = 15 ug. If an S/E of 50 is chosen for that sample, then 0.3 ug (15 ug ? 50 = 0.3 ug) of proteolytic enzyme should be added to the (15 ug total) sample.
Example: Chose S/E of 20 and 50 as starting points.
Determine or estimate the protein concentration of the unknown. If it is not known, try adding 0.3 ug of trypsin to the unknown sample, and 0.15 ug trypsin to a second sample that contains the same amount of unknown protein as the first sample.
- Promega trypsin stock solution preparation
Sequencing grade modified trypsin, catalog # V5111, 5 x 20 ug, is recommended (or an equivalent product). The enzyme is lyophilized. Solubilizing buffer is sold with the enzyme, in a separate tube.
Note: Modified trypsin is more resistant to autolytic digestion from reductive alkylation, when compared to non-modified trypsin, and is also affinity purified to remove a low-level contaminating protein, pseudotrypsin.
Promega product: ?20 ug aliquots of lyophilized protein (thaw on ice)
Solubilizing buffer: 50 mM acetic acid (thaw at RT then place on ice)
Example 0.3 ug/ul stock: (A different stock concentration might be more suitable; determine desired stock concentration according to the details listed in Step 3.) Pipette 65 ul of solubilizing buffer into a 20 ug aliquot of lyophilized trypsin. Be sure to wet the entire inside surface of the vial of lyophilized material, which entails aspirating the buffer and expelling, gently, multiple times, after initially pipetting the solubilizing buffer into the tube. After these steps, spin briefly in a microfuge for about 5 ? 10 seconds; place on ice.
- Protein sample/substrate/unknown
If the substrate is lyophilized prior to digestion, dissolve the target protein in 50 ? 100 ul of buffer (see step 2 for buffers).
If the protein is solubilized already, use an aliquot of 50-100 ul for the digest, for example. (If the protein concentration is known then an appropriate volume of sample can be determined according to step 3. )
Add (high purity) acetonitrile to the sample for a final concentration of 10% (v/v). Acetonitrile serves to help solubilize highly hydrophobic peptides that are generated during the digest. Trypsin is tolerant to 10% acetonitrile.
Determine the pH of the substrate solution. It must be in the range of 7.5 ? 9. To measure pH conveniently use colorpHast pH test strips 7.5-14 from EM Science (part #9587-3), for example, which have pH units in increments of 0.5 pH units.
Measure pH by pipetting 1 or 2 ul of sample onto one or two squares of the pH test strip. Match the color on the strip to the colors on the pH test strip box and determine pH. (Do not pipette from the sample tube after touching the pipette tip to the pH test strip squares, which contain chemicals. )
If the pH of a sample is lower than 7.5, add a small volume (2 ? 10 ul) of a concentrated buffer solution (0.1 M, for example), mix gently, spin for 5 ? 10 seconds, and check pH again. Repeat if pH is not in the appropriate range.
- Control protein- Ubiquitin (or chose another suitable protein)
Prepare an aqueous stock solution of ubiquitin, Sigma catalog number U-6253, at a concentration of 8. 6 mg/ml, 100 pmol/ul, for example (chose any appropriate concentration).
Prepare a ubiquitin working solution of about 86 ug/ml, 10 pmol/ul (ubiquitin MW = 8565 Da) in digestion buffer (see Step 2). Add acetonitrile; check the pH, be sure it is within 7. 5 ? 9.0 units.
- Add trypsin to samples
Add trypsin to the substrate and control samples in a very small volume (preferably 1-5 ul, therefore prepare stock trypsin solution accordingly). The pH of the trypsin solution is very low (the solubilizing buffer is acetic acid) therefore it is beneficial to add the trypsin in the smallest volume possible so that the pH does not change.
Gently tap tube, mix in microfuge for 5-10 seconds.
Place tube in heating block overnight/16 hours at 37 ºC.
Store trypsin at ?20 or ?80 ºC. Only 1 or 2 freeze/thaw cycles are recommended. The resonstituted enzyme is theoretically stable for 2 ? 3 months at ?20 ºC. The lyophilized powder is stable for 12 months at ?20 ºC.
- Stop the reaction
Remove samples from heating block. Allow the sample to cool (at RT or briefly at 20 ºC). Briefly spin in microfuge.
Add 1 ? 5 ul of high purity neat (concentrated) glacial acetic acid. Tap vial, spin in microfuge for about 5 ? 10 seconds, check pH with pH strips (for example, colorpHast pH test strips 2.0 ? 9.1 from EM Science, catalog #9578-3). Ensure that pH is at 3.0 (the lowest possible pH attainable with acetic acid).
Immediately store samples at -20 or -80 ºC or process for MALDI or other analysis.
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