Kunxin and Tamara' s Tryptic or Chymotryptic Peptide Mapping Protocol
This specfic protocol is the latest incarnation of peptide mapping procedures that have been developed here in the TVL/MBVL of the Salk Institute over the last 20 years. Wade Gibson developed the first peptide mapping protocol. This was improved by Karen Beemon and Tony Hunter. Tamara Hurley and Kunxin Luo adapted it to proteins transferred to filters.
1. Immunoprecipitate the protein and run it on a preparative gel. Scale up your immunoprecipitation so that you have at least 500 cpm. Transfer the protein to nitrocellulose, Immobilon or Nylon according to Kamps's Western Protocol. Tryptic digestion can be done with either NC, Nylon, or Immobilon. Nitrocellulose seems to give more reproducible yields of peptides.
Transfer of the protein to a filter is much superior to homogenization of the prep gel and elution. Although some people have concern about inefficient transfer of large proteins and loss of hydrophobic peptides on the filters, we have had good success with essentially every protein we have studied.
As a general principle, it is valuable to work fast and keep the filter damp. It is generally thought that the longer the protein remains bound to the filter, the lower the yield.
2. Rinse blots with water after transfer. Wrap the membrane in Saran wrap to keep the membrane wet during exposure. Try to keep the membrane damp throughout the procedure. If it dries out, you can rewet NC with water, or Immobilon with methanol and then water, but your recovery will probably go down.
After exposure, cut out the membrane piece of interest and soak it immediately in 0.5% PVP-360 (Polyvinylpyrrolidone, MW360) in 100mM acetic acid for 30 min. at 37°C. If you don't do this, your yield will be poor. This may result from the protease adsorbing onto the filter.
3. Aspirate the liquid. Wash the membrane extensively with H2O
(5 X 1ml). Then wash with freshly-made 0.05 M NH4HCO3 once or twice.
4. Digestion: Add 150 -200 microliter fresh 0.05 M NH4HCO3 (enough to cover the piece of membrane). Start the digestion by adding 10 micrograms trypsin or chymotrypsin (Stock is 1 milligram/ml in 0.05 M NH4HCO3 stored at -70°C) . Mix by pipetting up and down and incubate at 37°C for 2 hr.
5. Vortex the sample and add another 10 micrograms of enzyme. Incubate for 2 hr at 37°C.
6. Add 300 microliters (or more) H2O to each sample and spin in microfuge for 5 minute. Transfer the supe to new tube or remove the membrane. Dry on the speed vac. It takes about 3-4 hours.
7. Oxidation: The phosphorylated tryptic peptides of p56lck do not contain Met or Cys and oxidation is therefore not necessary. If the peptides you're examining contain Met or Cys, or if you don't know, you should oxidize them with performic acid.
To make performic acid, mix 1 vol. fresh 30% H2O2 with 9 vol. 98%-100% formic acid. Incubate the mixture at room temp. for 1 hr to allow performic acid to form. Cool on ice. The newly-made performic acid can be stored on ice for several hours before use.
Resuspend the dried peptides in 50 microliters performic acid by vortexing or pipetting up and down. Incubate on ice for 2hr. (1hr is probably enough. Just be consistent).
8. Add 1ml H2O to the tube. Dry on Speed-vac.
9. Resuspend the sample in 10 microliters H2O for an 8.9 map (or appropriate electrophoresis buffer if you're using another buffer for electrophoresis). Load 1 to 2.5¨microliters on the 0.1 mm thick, 20 cm x 20 cm cellulose thin layer plate from EM. The amount to load depends on how many counts you have, but you should not load more than 30% of the sample. More than that will cause the sample to smear in the electrophoretic dimension.
10. The location of the origin depends on the pH of the electrophoresis buffer. Peptides are generally positively charged at pH 1.9 or 4.72 and the origin is therefore displaced toward the positive electrode when using these buffers. As diagrammed above, the origin for pH 1.9 or pH 4.72 is 5 cm from the left (positive) edge and 2.5 cm up from the bottom.
The charge of phosphopeptides at pH 8.9 is less predictable and the origin for these maps is usually placed in the middle of the plate, 2.5 cm up from the bottom.
For phosphorylated Lck, run pH 8.9 map at 1KV for 27 min. in the 1st dimension.
11. It is important to run marker dyes. The green dye is a 1:1 mixture of Xylene cyanol FF and epsilon-DNP lysine in pH 4.72 buffer. It should be spotted in a position equivalent to that of the sample, but in a portion of the plate that will not contain peptides.
12. After electrophoresis, air dry the plate. It is thought that drying in the oven will cook the peptides irreversibly to the cellulose.
13. Then carry out chromatography. Here too you should use the green marker dye. Spot it off to the side, the same distance up from the bottom as the sample.
For phosphorylated Lck, do chromatography in Phosphochromo buffer .
Recently, chromatography has taken as long as 12 hours. It seems to vary from batch to batch of plates. All you can do is watch the plates and pull them all when the first has its buffer front approximately 1 cm from the top.
14. Then mark the plate with radioactive ink. It is useful to put marks on that can be used to figure out where the origin was and where the marker dyes ran. Don't mark the origin itself.
Usually, 3-4 days exposure with a screen and flashed film is necessary to detect samples containing 100-200 cpm of 32P.
It's sensible to make 2L of the electrophoresis buffers. The volume of chromatography buffer is dictated by the size of your tanks. You need enough to give a depth of approximately 0.5 cm in the bottom of the tamk
For 2 liters n-Butanol 100 ml Pyridine 50 ml Acetic acid 50 ml H2O 1800 ml
The pH should be adjusted to 4.72 using either acetic acid or pyridine. Note that the recipe in Wade's paper is wrong.
Ammonium carbonate 20 g H2O 2 L
The pH should be adjusted to 8.90 with either NH4OH or CO2
For 2 liters 88% Formic acid 50 ml Acetic acid 156 ml H2O 1794 ml
Don't use the 98% formic acid and don't adjust the pH.
n-Butanol 97 volumes Pyridine 75 volumes Acetic acid 15 volumes H2O 60 volumes
n-Butanol 75 volumes Pyridine 50 volumes Acetic acid 15 volumes H2O 60 volumes
If you want to read or cite the papers describing this technique, the appropriate references are:
Gibson, W. (1974) Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis.
Beemon, K., and Hunter, T. (1978) Characterization of Rous sarcoma virus src gene products synthesized in vitro. J Virol. 28(2):551-66. [abstract]
Luo, K., Hurley, T. R., and Sefton, B. M. (1990) Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene 5(6):921-3. [abstract]