Detection of Signal Transduction Protein Binding
Anti-phosphorylated peptide antibodies have become very useful reagents in the study of cell signal transduction. The signal transduction process involves a series of specific protein phosphorylations, each phosphorylated sequence providing a binding moiety and activation for proteins involved in the next stage in the cascade of protein binding and phosphorylation.
Specific antibody probes that are able to identify which of the many proteins in the signal transduction path are phosphorylated or have been de-phosphorylated help in the understanding of cell regulation, growth and apoptosis. The development of specific kinases inhibitors provides yet another target for the development of anti-cancer drug therapy, and indeed one of the most recent and most successful treatment for myoloid leukemia is a drug called “Glivac” a tyrosine kinase inhibitor.
Selection of peptide sequences
With the aim of identifying binding sequences in the phosphorylation cascade (Ward et al 1996), illustrated the use of the Multipin approach to identify all the specific peptide phosphorylation sites of a selected signal transduction protein. The study illustrated yet another application of the Geysen approach to systematic epitope mapping (Geysen et al 1984).
Where the specific phosphorylation sites of a protein are known or are being investigated, the challenge is to make an anti-peptide antibody, perhaps to both the phosphorylated and non-phosphorylated peptide sequences, that can be used as probes to identify the state of phosphorylation of the specific site on the protein being studied.
Factors involved in the production of Anti-phosphorylated peptide antibodies
Length of peptide
From several studies (Atassi 1975, Geysen et al 1988) it was concluded that around 5 residues are implicated in linear epitopes. Although it is possible to raise antibodies to shorter sequences or indeed to single residues,(Shisheva et al 1991), the shorter the peptide used as an immunogen the more likely the anti-peptide antibodies generated will cross-react and bind to other related proteins. As most protein phosphorylation sites are likely to be within a protein sequence and not at the very N- or C- termini, it would be wrong to conclude that simply using a 5-mer peptide coupled to an immunogenic carrier protein as an immunogen would generally be the best approach to raising useful anti-phosphorylated peptide antibodies, i.e. that are able to bind to the target peptide sequence in the protein. As a general observation when using short peptides as immunogens, antibodies are frequently generated which involve the free-end of the peptide. For example, if a short peptide, of the format, Amino-X1X2YpX4X5X6-Carrier Protein, where Xn represents the specific amino acid residues and Yp = phosphorylated tyrosine, then anti-peptide antibodies are likely to be generated that are specific to the amino terminus Amino-X1X2Yp…. This property of short peptides as immunogens can be used to great advantage where the aim is to create antibodies that are specific to the free ends of proteins. An interesting additional application of these free-end specific antibodies are as probes to detect proteolytic cleavage products.
Antibody Generation
As short peptides are generally not immunogenic in their own right it is necessary to couple them to immunogenic carrier proteins. To facilitate this coupling, a cysteine residue is usefully incorporated at either the N or C-terminus of the peptide and reacted to conjugate the peptide to an immunogenic carrier protein. (Lee et al 1980).
Generally, where the aim is to produce anti-phosphorylated peptide antibodies that can be used to detect the phosphorylated sequence within a protein, then it is better to use a longer peptide as the immunogen. We have found that a 10-15 residue peptide, where the phosphorylated residue is centrally located within the peptide sequence is frequently suitable.
No special immunization protocols are required to generate anti-phosphorylated peptide antibodies. Typically, animals of choice are immunized twice, several weeks apart. The first immunization is with an emulsion of the peptide conjugate with Complete Freunds adjuvant, the second using Incomplete Freunds adjuvant. Potent anti-peptide sera are obtained after several weeks (Palfreyman, et al 1984) It is important however to understand that other antibodies specific for non-phosphorylated peptide may also be generated. As the length of the immunizing peptide increases the chance that these antibodies will be generated also increases. To obtain the most complete information on the specificity of anti-peptide antibodies that have been generated, epitope mapping using a set of overlapping peptides is convenient. Although not involving a phosphorylated-peptide as an immunogen Fig. 1 shows the results of testing a rabbit antiserum to the peptide EIPEPYVWDESFRVC (from myohemerythrin) on a set of overlapping pentapeptides homologous with the immunizing sequence. The immunogenic conjugate was prepared by coupling the peptide to Keyhole Limpet Hemocyanin via the C-terminal Cysteine. The results clearly identify two linear epitopes within the sequence, the implicated epitopes being centered on tetrapeptides IPEP and DESF.
The understanding that other specific anti-peptide antibodies, not involving the phosphorylated residues, may be generated is crucial to how the phosphorylated peptide antisera are used, and to the interpretation of results obtained using these antibodies.
Testing Phosphorylated-Peptide Antisera
Although not as thorough as a full epitope mapping, it is useful to determine the relative titers of sera to determine how specific they are for both phosphorylated and non-phosphorylated version of the peptide.
This is conveniently done using an enzyme linked immunosorbent assay (ELISA) where the sera are titrated on microtiter plates coated with non-phosphorylated-peptide and phosphorylated-peptide. Where the titer of specific anti-phosphorylated peptide antibodies are very much greater than the titer to the non-phosphorylated peptide then the sera may be used at an appropriate dilution, i.e. without further processing, to probe for the phosphorylated protein. Even though antibodies specific for the non-phosphorylated peptide may be present, any low level binding would not interfere with interpretation of the data.
Where the level of antibodies to the non-phosphorylated peptide is relatively high, or perhaps even greater that the level of anti-phosphorylated peptide antibodies, then using the antisera at any dilution would result in ambiguous results, i.e, both the phosphorylated and non-phosphorylated protein sequence would be detected.
Phosphorylated peptide antibody enhancement
Where a significant level of antibodies specific to the non-phosphorylated peptide is present in the antisera, then useful anti-phosphorylated peptide antibodies can still be obtained using an enhancement process.
To achieve this, the non-phosphorylated analog of the peptide used for the immunization is coupled to Thiopropyl-Sepharose 6B (Pharmacia product code 17-0420-01) using the available cysteine residue, following the manufacturers instructions.
The resultant gel is incubated with aliquots of the antisera to absorb antibodies specific to the non-phosphorylated peptide (See Figure 2).
The resultant antiserum will have an enhanced specificity for the phosphorylated peptide sequence.
It will still be desirable to test the resultant enhanced antisera to confirm the level of enhancement achieved, and establish the dilution to be used to probe for the phosphorylated protein.
Affinity purification of anti-phosphorylated peptide antibodies
Affinity purified antibodies are antibodies which have been adsorbed onto the specific peptide, and after washing away any unadsorbed protein, the specific antibodies are eluted from the peptide (adsorbent), usually by application of a general technique such as low pH elution. Although enhanced antisera may be suitable for many applications, there may circumstances where affinity purified antibodies are required. For example, affinity purification may be required where an unacceptable level of background binding is observed using the enhanced serum, possibly caused by other antibodies present in the serum; or when absence of other proteins is important, such as when direct labeling of the antibody is required.
It would be a mistake to use the phosphorlyated version of the peptide, coupled to a gel, to affinity purify antibodies directly from antiserum. Eluted antibodies from this gel may be free from other extraneous antibodies and serum components, however antibodies specific for both the phosphorlyated and non-phosphorlyated peptide sequence would be present (see Figure 3a and 3b).
To produce affinity purified antibodies that are specific to the phosphorlyated peptide only, it is necessary to first perform the enhancement procedure to remove antibodies from the serum that are specific to the non-phosphorlyated peptide. If required, antibodies specific for the non-phosphorlyated peptide can also be obtained, by elution of antibodies off the non-phosphorlyated peptide–gel.
References
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- Geysen, H.M., Meloen, R.H. and Barteling, S.J. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA. 81, 3998.
- Geysen, H.M., Mason, T.J. and Rodda, S.J. 1988. Cognitive features of continuous antigenic determinants. J. Molecular Recognition 1, 32.
- Gras-Masse, H.S., Jolivet, M.E., Audibert, F.M., Beachey, E.H., Chedid, L.A., Tartar, A.L. 1986. Influence of CONH2 or COOH as C-terminus groups on the antigenic characters of immunogenic peptides. Mol. Immunol. 23, 1391.
- Lee, A.C.J., Powell, J.E., Tregear, G.W., Niall, H.D. and Stevens, V.C. 1980. A method for preparing b-hCG COOH peptide-carrier conjugates of predictable composition. Mol. Immunol. 17, 749.
- Palfreyman, J.W., Aitcheson T.C., Taylor, P. (1984) Guidelines for the production of polypeptide specific antisera using small synthetic oligopeptides as immunogens. J. Immunol. Methods 75; 383-393.
- Shisheva, A., Leitner, O. and Shechter, Y. 1991. A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphorylatedtyrosine. J. Biochem. Biophy. Meth., 23: 307-314.
- Ward CW, Gough KH, Rashke M, Wan SS, Tribbick G, Wang J. 1996 Systematic mapping of potential binding sites for Shc and Grb2 SH2 domains on insulin receptor substrate-1 and the receptors for insulin, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. J Biol Chem 1996 Mar 8;271(10):5603-9