Determination of phosphorylated amino acid positions in peptides

Summary

If the sample volume is insufficient for direct sequencing, the peptide can be degraded by manual Edman degradation to determine the location of the phosphorylated amino acid in the peptide. Source: Compact Molecular Biology Laboratory Guide (5th Edition)

Operation method

Basic program Manual EDMAN degradation

Principle

Degradation of serine or threonine phosphate releases serine or threonine and free phosphate, while degradation of tyrosine phosphate releases anilinothiazolone, a derivative of tyrosine phosphate.

Materials and Instruments

Eluted phosphopeptides (see Supporting Scheme 1)
5% (V/V) phenyl isothiocyanate (PLTC) dissolved in pyridine 10:1 (V/V) heptane/ethyl acetate-10 parts of heptane mixed with 1 part of ethyl acetate 2:1 (V/V) heptane/ethyl acetate-2 parts of heptane mixed with 1 part of ethyl acetate 100% (m V) trifluoroacetic acid (TFA) Electrophoresis buffer (pH 1.9 200~500 cpm 32P (prepared by diluting 32P orthophosphate with deionized water) or 2 mg/ml PTH-phosphotyrosine (see Supporting Solution 1) Electrophoresis buffer (pH 1.9 200~500 cpm 32P (prepared by diluting 32P orthophosphate with deionized water) or 2 mg/ml PTH-phosphotyrosine)
Microcentrifuge tube 45℃ Water bath Scintillation counter for Cherenkov counting TLC plate (20 cm × 20 cm 100 μm cellulose) 65℃ Oven or fan

Move

1. Determine the number of cycles from the list of candidate peptides.

Specify the number of cycles as ^ The starting volume for each cycle is 20 μl.

2. dissolve the eluted peptide in 20 μl of deionized water in a tube called a reaction tube (microcentrifuge tube).

3. take a 20/(x+1) μl portion of the sample into a new tube; this is the starting sample material and is stored at 4°C. 4. replenish with deionized water.

4. refill the reaction tube with deionized water to bring the total volume back to 20 μl. begin counting the samples at this point:

4.1 Ensure that the desired amount of cpm is actually removed from the starting sample (as starting sample material);

4.2 Detect the cpm at the beginning of each cycle.

5. Add 20 μl of 5% (V/V) phenyl isothiocyanate (dissolved in pyridine) to each reaction tube, mix well by vortexing and shaking, briefly centrifuge to collect liquid to the bottom of the tube, and incubate for 30 min at 45°C. 6. Add 200 μl of 5% (V/V) phenyl isothiocyanate to each reaction tube.

6. Add 200 μl of 10:1 heptane/ethyl acetate to each tube and vortex for 15 s. Separate the two phases by centrifugation for 1 min at full speed.

The pyridine will go into the organic phase (upper layer). 7.

7. Using a pipette with a plastic tip, carefully remove the upper organic phase. Extract the aqueous phase (lower layer) again with 10:1 heptane/ethyl acetate as in step 6. 8.

8. Extract the aqueous phase twice with 2:1 Heptane/Ethyl Acetate as in Step 6. 9.

9. Freeze the aqueous phase on dry ice and lyophilize in a SpeedVac evaporator.

10. Redissolve the dried sample in 50 μl of 100% trifluoroacetic acid (TFA) and incubate at 45 °C for 10 min. 11.

11. Lyophilize the sample in a SpeedVac evaporator. 12.

12. Count the sample by Cherenkov counting.

The measured cpm should be the same as the value at the beginning of the cycle (i.e., step 4). 13. Add 20 cpm to the reaction tube.

13. Add 20 μl of deionized water to the reaction tube, vortex, and centrifuge briefly. Remove 20/x μl of the product of the first cycle for analysis. Remove the sample and store at 4°C along with the starting sample. 14.

14. Add additional deionized water to the reaction tube to bring the total volume back to 20 μl and start the second cycle. Repeat steps 5-12. 15.

15. After the second reaction, resuspend the remaining sample with 20 μl of deionized water and remove 20/(x -1) μl and transfer to a new tube as the product of the second reaction for analysis. Repeat steps 4 to 12. 16.

16. Continue repeating steps 4~12 until the desired number of reaction rounds has been performed.

For each new cycle, take 2 (x-y) μl of sample, where y equals the number of cycles minus 1. 17.

17. All samples are lyophilized in a SpeedVac evaporator. All final samples are counted using the Cherenkov counting method. 18.

18. If lyophilized, dissolve the samples in 5 μl of pH 1.9 electrophoresis buffer or deionized water. Centrifuge at maximum speed for 2 min to precipitate insoluble material.

If the volume of sample taken after each round of reaction is very small, steps 17 and 18 can be skipped and the sample can be upsampled directly onto a TLC plate.

19. Spot all samples analyzed for a given phosphopeptide at least 1 cm apart at a starting point on a vertical line in the center of the TLC plate (see Figure 17.9.5). 50-200 cpm [32P] phosphate or 1-2 μg PTH-phosphotyrosine (0.5-1.0 μl 2 mg/ml PTH-mercuric acid tyrosine reservoir), depending on the phosphoamino acid composition of the peptide under study, is used as a marker to spot at the starting point on the same vertical line. The sample is dispensed in drops of 1/3 to 1/2 μl at a time and dried before the next drop is added (see step 25 of Basic Scheme 1).

20. Wet the plate as shown in Figure 17.9.5. 21.

21. Prepare the HTLE7000 device for electrophoresis in electrophoresis buffer pH 1.9 at 1.0 kV for 25 min (see Basic Plan 1 and Figure 17.9.3). 22.

22. Allow the plates to dry (oven or fan at 65°C), label with radioactive or fluorescent markers, and expose the pre-sensitized film at -70°C with the aid of a sensitizing screen (radiographic autoradiography).

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