More information about phosphopeptides can be obtained by digestion with sequence-specific proteases or cleavage with site-specific chemicals. From The Compact Laboratory Guide to Molecular Biology (5th Edition)
Operation method
basic program
Materials and Instruments
Eluted phosphopeptides Move 1. Dissolve the eluted phosphopeptide in 50 μl of suitable buffer in a microcentrifuge tube and centrifuge briefly at the bottom of the tube to collect all of the solution. Add 1 μl of the peptide solution dropwise to pH paper to check the pH and ensure that the final pH is appropriate for the enzyme activity. If the pH of the solution changes drastically with the addition of the peptide, adjust the pH before adding the enzyme. 2. 2. Remove a portion (usually 50%) of the sample as an undigested control and as part of the mix with the digested sample. 3. 3. add 1 to 2 μg of enzyme to the sample to be digested, vortex and oscillate, and centrifuge briefly to concentrate the sample to the bottom of the tube. 4. Place all tubes in a water bath at the appropriate temperature and incubate for at least 1 h. 5. Add a further portion of enzyme and continue to incubate. 5. Add another portion of enzyme and continue incubation for 1 h. 6. Add 1 portion of enzyme to each sample. 6. Add 1 μl of 2-mercaptoethanol to each sample and boil for 5 min to inactivate the enzyme. 7. Lyophilize the samples in a SpeedVac evaporator. 8. 8. Resuspend samples in 6 μl of electrophoresis buffer of appropriate pH and mix by vigorous vortexing to dissolve samples. Centrifuge at full speed to precipitate the undissolved material. 9. 9. Spot half of the undigested sample onto a TLC plate. The digested sample is divided in half and spotted onto two TLC plates, one of which is then spiked with the other half of the undigested sample. 10. Electrophoresis and chromatography are performed on the plates (Basic Scheme 1 steps 24 to 31). Choose the appropriate pH and time of electrophoresis and chromatography based on the position of the specific phosphopeptide being analyzed on the original plot, so that the peptide and its possible degradation products are as well separated as possible, while ensuring that small degradation products remain on the plate. Caveat When analyzing a mixture of digested and undigested samples the enzyme must be completely inactivated before spotting the sample onto the plate, otherwise the undigested sample will be digested very quickly when the two samples are mixed. Common Problems Preparation of the eluted phosphopeptides can be seen in the auxiliary protocol for the Come XX experiment. For more product details, please visit Aladdin Scientific website.
Digestive enzymes Digestion buffer 2-mercaptoethanol Electrophoresis buffer of suitable pH
Water bath TLC plates (20 cm × 20 cm 100 vinyl) at a temperature suitable for enzymatic digestion