Peptide Synthesis

Peptides are complex molecules and each peptide sequence is unique with regard to its chemical and physical properties. Peptides are synthesized by the solid-phase method using Fmoc chemistry on an Omega 396 multiple peptide synthesizer from Advanced ChemTech. The Core also uses an Applied Biosystems Model 431A Synthesizer for longer peptides. Synthesis is carried out at 50 µmole, 100 µmole or 250 µmole scales. On an average, a synthesis scale of 100 µmoles will yield about 50-70 mg of a 10-residue peptide and 100-150 mg of a 20-residue peptide depending on the substitution rating of the resin. The peptide synthesis service provides a complete spectrum of services that include, synthesis, cleavage, HPLC purification, peptide modifications, cyclization and mass analysis for quality assurance. Before submitting your peptide sequences to the Core, it is advisable to consider the following:

Every peptide sequence synthesized is a unique one. Some peptides sequences are difficult to synthesize although, we attempt to synthesize every peptide sequence.
Peptides with multiple cysteines, methionine, arginine and tryptophan residues can often be difficult to synthesize.
Avoid a proline residue at the C-terminal of the peptide.
The peptide will be de-protected and cleaved from the resin with 82.5-90% trifluoroacetic acid, partially purified, and lyophilized. The lyophilized material will be provided as a "crude" preparation. Turnaround time is about ten days to three weeks depending on the length of the peptide and purity level. When requested, the peptide will be purified further (see below).

Quality Control Check

Strict quality control in peptide synthesis is the most important part. Besides synthesis related problems, there are post-synthesis adduct formation and modification problems associated with peptides. An analytical-scale reverse-phase HPLC chromatogram and the mass analysis by either MALDI or electro-spray mass spectrometry will be provided for every peptide to ensure the highest quality of synthetic product. The Core will also get the amino acid analysis performed from an outside facility if so requested.

Modification of Synthetic Peptides

The core laboratory routinely synthesizes peptides with the following modifications: peptides containing unusual or modified amino acids (e.g., phospho-Tyr, phospho-Ser/Thr, D-amino acids or other analogs), biotinylation at N- or C- terminals, fatty acid conjugation (butyric, hexanoic, myristic, palmitic), fluorochrome conjugation (fluorescein, rhodamine, phycoerythrin) N-acetylation, peptides amides, and peptide cyclization via s-s bonds.

Peptide Purification

A successful synthesis is dependent on the sequence of the peptide. Likewise the coupling efficiency of amino acids in the sequence and ultimate purity of the synthesized product is also related to the sequence. Impurities such as salts, deletion peptides (created due to incomplete coupling), protecting groups and modified peptides can range from 10-50% by weight. It is recommended that all the peptides should be purified prior to use. Purification of synthetic peptides is carried out on a semi-preparative Waters HPLC system using a RP C-18 column (2.2 cm x 25 cm). Fractions containing the peptide will be collected and lyophilized. In general, 50 mg to 70 mg of crude material will be injected at a time to obtain 20 mg to 50 mg of purified peptide at a minimum purity of 95%. The actual purity obviously depends on how conservatively the fractions are pooled. Users need to specify what level of purity is required.

Mass Spectral Analysis

MALDI-TOF (Matrix Assisted Laser Desorption Ionization –Time Of Flight) mass spectrometry is available for peptide and protein up to 50 KDa. Mass accuracy is generally 0.03% and typical sensitivity is 1 microgram depending on the peptide.

Peptide Storage

Peptides are provided as lyophilized product that should be stored as dry powder at -20°C to -70°C in a desiccator, if possible. After lyophilization, peptides retain significant amounts of water. Peptides are slowly degraded particularly the cysteine-containing peptides are oxidized over time at -20°C. The water content of a given peptide powder varies depending on sequence of the peptide, storage conditions, handling, and other factors. Some peptides are very hygroscopic. A lyophilized peptide also contains counter-ions that are paired with charged residues (e.g., acetate or trifluoroacetate for positively charged residues). The presence of counter-ions cannot be avoided because of ion pairing. Water and counter-ion content in a given peptide product vary widely and combined together may be as high as 30%. The exact concentration of the peptide in the crude product can be determined by amino acid analysis using a small aliquot from the crude peptide solution. The volume of this aliquot must be accurately measured and recorded because this value is necessary for calculating the concentration of the stock-solution. Peptide solution is much less stable than the lyophilized products. The peptide solution can be stored with the following guidelines:

The peptide solution should be stored at -20°C in small aliquots. The stock solution should be aliquoted upon arrival to prevent degradation caused by repeated freezing and thawing.
Pass the peptide solution through a sterile filter before storing to prevent bacterial contamination
Maintain peptide in an oxygen-free environment as peptides having cysteine, methionine, trypophan, glutamine, and asparagine residues are susceptible to oxidation and have reduced self-life.

Reconstitution of Lyophilized and Frozen Peptides

When using frozen peptides, the vial or tube should be allowed to warm to room temperature in a desiccator containing fresh desiccant before opening. Failure to do this can cause condensation to form on the peptide when the vial is opened and will affect the stability of the product. The best solvent to reconstitute the peptide is DMF (up to 30%), added drop wise until the peptide dissolves.

Peptide Conjugation to a Carrier Protein for Immunization

The sequence of an antigen peptide should be designed such that the peptide can be attached to a carrier-protein (e.g., keyhole limpet hemocyanin, ovalbumin, bovine gamma-globulin, or bovine serum albumin). Peptides can be conjugated to these carrier reagents through either its amino- or carboxyl-terminal ends. This type of cross-linking to C- or N- terminal can be performed via a pre-activated carrier protein. The commonly used cross-linking reagents are: MBS (m-maleimidobenzoyl-N-hydrosuccinamide) cross-linker to C, K or free amino groups; EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride cross-linker to Asp, glu or a carboxy terminus. The extent of coupling will be estimated by amino acid analysis.

The Multiple Antigen Peptide System

The Multiple Antigen Peptide (MAP) concept was introduced in 90’s to avoid chemically undefined entity of an antigen-carrier system. The MAP system utilizes a peptidyl core of three or seven radially branched lysine residues, on to which the antigen sequences of interest can be built using standard solid-phase chemistry. The lysine core yields the MAP bearing four or eight copies of the peptide epitope (high molar ratio), depending on the inner core that generally accounts for less than 10% of total molecular weight. The MAP system does not require a carrier protein for conjugation. The high molar ratio and dense packing of multiple copies of the antigenic epitope in a MAP has been shown to produce strong immunogenic response. Synthesis of a multiple antigenic peptide is carried out at a 0.1 mmol scale. This ensures maximum coupling yield during synthesis and, hence, optimizes the preparation.