C.B.S. Scientific HTLE-7002 - Manual (Page 12)

C. B. S. œ Scientific 12 HTLE-7002
B. Precipitation—continued
If your samples are P
32
-labeled then determine Cherenkov counts remaining and determine
the percent recovery. Your yield should be about 70% of your starting counts unless your
protein was unusually large or you used a very high concentration acrylamide gel during the
purification. If your recovery was low, check both the TCA supernatant and ethanol/acetone
wash for residual counts. If the majority of your counts remained in the TCA supernatant, try
spinning again. Replace it on ice for an additional 1-4hr and repeat the centrifugation and
washing steps.
C. Proteolytic Digestion and Oxidation
For primary digests of the TCA precipitated protein usually only trypsin or chymotrypsin are
capable of digestion to completion, although a variety of proteases will yield reproducible
fingerprints such as S. aureus V8 protease, thermolysin, P. fragi protease and proline-
specific endopeptidases. The example treatment used below will illustrate the use of trypsin,
but any of the enzymes listed above could be used instead using the same enzyme
concentration and sample treatment. Prepare stocks of TPCK-treated trypsin (Worthington
Enzymes) and other enzymes in 0.1 mM HCl at a concentration of 1.0 mg/ml and store in
small aliquots in liquid nitrogen.
Beginning with the washed and dried TCA precipitate one has several options: I) proceed
directly with trypsinization, ii) use the entire sample to determine phosphoamino acid content
(see below) or iii) use portions for both tryptic peptide analysis and for phosphoamino-acid
analysis. For either direct trypsinization or for combined tryptic and phosphoamino acid
analysis, dissolve the precipitate in 50-100
μl of ice-cold performic acid and oxidize at 0°C for
60 min (do not warm sample). Remove a small portion of the dissolved sample at the end of
this period if phosphoamino acid analysis is needed (see below). After complete oxidation,
add 400
μl of deionized water to dilute the performic acid and freeze the sample.
Since performic acid can cleave peptide bonds at higher temperatures it is important to both
dilute the sample in water and freeze as soon as the oxidation step is finished. The frozen
sample is usually lyophilized to completion. This usually takes 3-4 hr but can go overnight for
convenience. A “Speed-Vac” from Savant is very good for this process because the sample
is concentrated in the bottom of the tube. The sample should appear as white and stringy
material like a miniature cottonball, not as a hard pellet.
When lyophilization is complete, resuspend the oxidized protein pellet in about 50
μl of
ammonium bicarbonate, pH 8.0-8.3. The ammonium bicarbonate solution prepared on the
previous should have the correct pH. Vortex well and spin the tube briefly to bring all the
liquid to the bottom. Of the tube. For standard trypsinization, add 10
μl of a stock 1.0 mg/ml
solution of TPCK-trypsin (10
μg), close the lid tightly and incubate for 3-5 hr (or overnight) at
37
°C. At this time vortex the digest well (at least 30-40sec), centrifuge to condense the
sample as before, then add an additional 10
μl of stock trypsin and incubate again for 3-5hr at
37
°C.
Incubation periods lasting 2-3hr each should be sufficient, and one could justify using less
trypsin (1-2
μg total). However we follow the original protocol and have not modified it,
because it is very reproducible and seems to solubilize the maximum number of counts from
the precipitate. Others have tried short, sequential additions (repeated 5 times) of 0.1
μg of
trypsin for 30-60min. This may be useful when trying to obtain complete tryptic digests while
eliminating any contaminating chymotryptic activity.