- Design Considerations
- Synthesis, Cleavage, and Extraction
- Purification of Peptides
- Quality Control
- Peptide Storage
Synthetic peptides have a wide variety of uses ranging from structure-function analysis of sites within a protein, binding assays, receptor agonists/antagonists, to immunogens for the production of antisera. Regardless of the specific application, all that is required from the investigator to initiate peptide synthesis is the sequence of the desired peptide and an account number. While we cannot give a 100% guarantee that we will be able to synthesize a particular sequence (see next paragraph) we will at least attempt to synthesize virtually any sequence. The Peptide Synthesis Service provides a complete range of services including cleavage, HPLC purification, quality assurance analysis and a variety of amino acid derivatization/labeling techniques.
Although there have recently been significant advances in solid phase peptide synthesis, most notably the virtually universal transition from t-Boc to FMOC based chemistries, peptide synthesis still remains somewhat of an art, definitely not as routine as oligonucleotide synthesis, despite what instrument manufacturers may wish us to believe. In the past, particular amino acids or combinations of amino acids posed significant synthetic obstacles, as did longer peptides. The new chemistries have shifted the synthetic problems to primarily sequence based ones. While this has allowed the more routine synthesis of short peptides, and increased the potential for successful synthesis of longer peptides, prediction of problem regions is still often elusive. This makes stringent quality assurance measurements for every peptide synthesized at the PDTC an essential part of the synthetic process. Each peptide delivered to an investigator is fully analyzed by analytical reversed phase HPLC and liquid chromatography mass spectrometry.
One of the most often asked questions is what region of a protein to synthesize to produce anti-peptide antisera. Although there is no one correct answer for any protein, there are a number of computer programs derived from several publications that provide good approximations for immunogenic regions within proteins (1-4). These are available from the Rockefeller University Computer Services in the Smith Hall User Area. Please refer to them for additional information on these programs.
Prior to finalizing your peptide sequence please consider the following points:
- More than 8 arginine residues in a single sequence can cause incomplete cleavage problems and problems with severe adduct formation in crude peptides.
- Sequences that have repeating amino acids in multiple locations have displayed poor recovery due to poor coupling yields. For example the following sequence is very difficult to synthesize due to the numerous occurrences of repeating amino acid pairs: H2N-AARTTHGLlDDDERR-COOH
- High occurrence of Ala, Leu, Ile, Val, Glu, and Ser in the last several residues (from the C-terminus) can lead to problems due to aggregation of the peptide during the initial synthesis work.
- Side reactions can occur during the synthesis if the following amino acid pairs are present in the sequence: Asp-Ala, Asp-Gln, Asp-Asn, Asp-Gly.
- Avoid GLN or PRO as N-terminal residues as they can result in pyroglutamyl formation.
- If the peptide is to be conjugated to another carrier protein, peptide or reagent, it is preferable that the coupling be done through the N- or C- terminus. Cross-linking through an N- or C- terminal Cys to a preactivated carrier protein is by far the most common and easiest way to conjugate a peptide. Below are listed some of the more common crosslinking reagents and the predominant amino acids crosslinked by them:
- BDB (bis-diazobenzidine): TYR coupling
- MBS (m-maleimidobenzoyl-N-hydrosuccinamide): Cys, Lys, or Free NH2 groups
- EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride): Asp, glu or a C-terminus
- By far the most difficult peptides to successfully deliver with adequate yields are very hydrophobic peptides, i.e. ones that contain a high proportion of hydrophobic side groups and few or no charged ones. These peptides can be both difficult to synthesize as well as difficult to deal with during the cleavage and purification process. Extremely hydrophobic peptides are especially difficult to purify using the reversed phase HPLC techniques that are normally used.
Above all please talk to us about your peptide. Advances in this field occur regularly. Peptides that were impossible to make two years ago may be quite simple to make today. Side reactions can be minimized through the use of special amino acid derivatives, and may not pose as great a problem as anticipated from an examination of the sequence. Rarely is a sequence not accepted for synthesis.
Oligopeptides are synthesized with an Applied Biosystems Model 430A instrument using FMOC based chemistry and uronium salt type activation methodologies, such as HBTU/HOBt/DIEA activation. This chemistry provides for faster synthesis cycles, better coupling kinetics, and greatly improved coupling efficiencies. These improvements result in faster turnaround times and lower prices for peptides. Additionally, FMOC peptides can be cleaved using a trifluoroacetic acid/cation scavenger cocktail, obviating the need for costly and time consuming HF or TFMSA cleavages required for t-Boc peptide synthesis. At this time t-Boc synthesis is not used for the production of peptides in the Peptide Synthesis Laboratory.
The Peptide Synthesis Laboratory routinely performs the following amino acid modifications:
- Biotinylation : either N- or C-terminus. N-terminal biotinylation is the preferred method because the yields are dramatically higher and the cost for reagents is much lower.
- Lipid conjugation: peptides can be crosslinked to preformed lipid vesicles and used as immunogens without adjuvants (5,6)
- Phosphorylation: the peptide lab specializes in synthesis of phospho- Ser, Thr, and Tyr containing peptides. Successful synthesis of peptides containing several selectively phosphorylated residues is possible.
- N-terminal Fluoresceination: labelling of the N-terminus of the peptide with FITC (Fluorescein Isothiocyanate).
- Mimetics: Unnatural and unusual amino acids and amino acid analogs can often be incorporated into a peptide. Examples include: ornithine, norleucine, L-malonyltyrosine (a non hydrolyzable analog of phosphotyrosine) (7) and blocking of the N-terminus with N-acetylglycine.
These modifications can have stringent requirements. Please call to make an appointment with a Peptide Synthesis Specialist to discuss your particular application and requirements.
Since the purity of the peptide obtained from the synthesis is sequence dependent, purification to >95% will be performed upon request. Major impurities can range from 10% to 50% by weight. They consist of small water soluble molecules, salts and protecting groups from the cleavage reaction, deletion peptides created due to incomplete coupling during synthesis, and modified peptides created during the cleavage. These species can be removed using reversed phase HPLC. We recommend that, under most circumstances, all peptides be purified prior to employing them in research studies.
Since the peptides synthesized by the PDTC often form the basis for months and years of research and publications, we strongly believe that stringent quality control is an essential component of the synthesis process. To ensure the highest quality of products we regularly and frequently perform preventive maintenance on our synthesizers and ancillary equipment. Additionally, prior to each synthesis run a full calibration check is performed on the synthesizer to make sure that the instrument is running within specifications.
Post-cleavage adduct formation, salt complexing, deletion peptides, and incomplete deprotection during cleavage, continue to be problematic for all peptide synthesis labs. As a measure of the quality of the peptides produced the Peptide Synthesis lab provides the following to investigators:
- Reversed-phase HPLC: Each peptide delivered is accompanied by an analytical RP-HPLC chromatogram of the delivered product. The gradient is run for sixty minutes with a 1% per minute slope, which provides peak resolution comparable or superior to standard traces provided by commercial labs. Four chromatograms at 220, 263, 280, and 300 nm are provided as a measure of the peptide compositional purity as well as an indication of the presence of incompletely deprotected species. Additionally, each 220 nm chromatogram is individually integrated by a peptide synthesis specialist to provide a quantitative measure of the peptide compositional purity.
- Mass Spectrometry: Mass spectrometric analysis of the delivered peptide is provided to the investigator. A Perseptive Biosystems Voyager MALDI-TOF system is used to perform the analysis. Although this method does not provide quantitative data, it does provide a reliable method for ascertaining the proper identity of the peptide.
Hydrolysis of a peptide bond is slow but does occur. Store peptides as dry as possible at -20C or -70C. Desiccate if possible. Remember that hydrophobic peptides bind avidly to glass. Cysteine containing peptides will oxidize over time, even when stored at -20C. Long term storage of peptides in solution is not recommended regardless of the temperature.
- Hopp, TP and Woods, KR (1981) Prediction of protein antigenic determinants from amino acid sequences. Proc. Nat. Acad. Sci., USA 78:3824-3828
- Hopp, TP and Woods, KR (1983) A computer program for predicting protein antigenic determinants. Mol. Immunol. 20:483-489
- Kyte, J and Doolittle, RF (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132
- Westhof, E, Altschuh, D, Moras, D., Bloomer, AC, Klug A, and Van Regenmortel MHV (1984) Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature 311:123-126
- Martin, FJ and Papahadjopoulous, D (1983) Irreversible coupling of immunoglobulin fragments to preformed vesicles. J. Biol. Chem. 257:286-288
- Hashimoto, Y, Sugawara, M and Endoh, H (1982) Coating of subunits of monoclonal IgM antibody targeting of the lipsomes. J. Immunol. Mets. 62:155-162
- Kole, HK, Akamatsu, M, Ye, B, Yan, X, Barford, D, Roller, PP, and Burke, TR Jr. (1995) Protein-Tyrosine Phosphatase Inhibition by a Peptide Containing the Phosphotyrosyl Mimetic, L-O-Malonyltyrosine. Biochem. Biophys. Res. Com. 209:817-821