Peptide Synthesis: Handling and Storage of Synthetic Peptides

1. How should I store and handle my synthesized peptides?

Peptides are shipped at room temperature and highly stable at lyophilized form in sealed vials. Peptides should not be stored in solution for long time storage.

Peptide storage guidelines: For longer storage, peptides should be stored in lyophilized form at -20°C or preferably at -80°C in a sealed container with desiccant in order to minimize peptide degradation. Under these conditions, the peptide can be stored for up to several years and the following activities can be prevented such as bacterial degradation, secondary structure formation, and oxidation.

Open the package: It is better to equilibrate the peptides to room temperature in a desiccator prior to opening and weighing. Failure to warm up beforehand can cause condensation to form (peptides tend to be hygroscopic) on the product when the bottle is opened and this will reduce the stability of the peptide products.

Weigh peptides: Weigh out you needed quantity of peptide quickly and store all unused peptide at -20°C or less. Sequences containing cysteine, methionine, tryptophan, asparagine, glutamine, and N-terminal glutamic acid will have a shorter shelf life than other peptides.

2. How should I dissolve peptides?

The solubility of a peptide is determined mainly by its polarity. Acidic peptides can be reconstituted in basic buffers and basic peptides in acids. Hydrophobic peptides or neutral peptides containing large number of polar uncharged amino acids or hydrophobic amino acids should be dissolved in a small amount of organic solvents such as DMSO, DMF, acetic acid, acetonitril, methanol, propanol or isopropanol and then diluted in water. DMSO should not be used if the peptides containing methionine or free cysteine because it may oxidize the side-chain functionalities.

It is necessary for you to test a portion of the synthesized peptide first before dissolving the entire peptide sample. You may need to test different solvents until you find an appropriate one. It is necessary to sonicate the peptide solution because sonication enhances solubilization.

1. If the peptide is acidic, add small amount of basic solvents such as 0.1M ammonium bicarbonate to dissolve the peptide and add water to the desired concentration. Acidic peptides may be soluble in PBS (pH7.4). Peptides containing free cysteines should be dissolved in degassed acidic buffers. The thiol moieties will be rapidly oxidized at pH>7 to disulfides.
2. If the peptide is basic, add small amount of acidic solvent such as acetic acid or trifluoroacetic acid. You may use 25% acetic acid to dissolve the peptide and add water to the desired concentration.
   
3. If peptides are still insoluble, add acetonitrile to 20 %(v/v), and sonicate the sample.
4. If, after sonication, the peptide has not dissolved completely, lyophilize and remove the volatile buffer solution such as water, acetic acid or acetonitrile. When the sample is completely dry, dropwisely add DMF or DMSO until the peptide dissolves. Slowly dilute the solution with water to approximately 10% (v/v) DMF or DMSO. Dilute the stock solutions to the same peptide concentration.
5. In order to prevent or minimize peptide degradation, store the peptide in lyophilized form at -20°C or preferably at -80°C. If the peptide is in solution, freeze thaw cycles should be avoided by freezing individual aliquots.

3. Can I predict if a peptide is soluble?

We cannot predict the solubility of a peptide in water by studying the structure. However, the ε-amino group of Lys and guanidine function of Arg are usually helpful for the solubility especially in short sequence. By contrast, the acidic peptides containing Asp and Glu tend to be insoluble in water but they can be easily dissolved by diluted ammonia and by basic buffers.

4. How should I choose the peptide purity for my research?

Crude peptides are not recommended for biological assays. Crude peptides may contain large amount of non-peptidic impurities such as residual solvents, scavengers from cleavage, TFA and other truncated peptides. TFA cannot be totally removed and peptide is usually delivered as TFA salt. If residual TFA is a problem for your experiment, we recommend other salt forms such as acetate and hydrochloride. These salt forms are usually 20-30% more expensive than the regular TFA salt. This is due to the peptide loss during the salt conversion and more raw materials are needed for this purpose.

LifeTein recommends the following peptide purity for your research:

>70% purity

• Peptide array
• Antigen for antibody production
• Competitive elution chromatography
• ELISA standard for measuring antisera titers

>80% purity

• Western blotting studies (non-quantitative)
• Enzyme-substrate studies (non-quantitative)
• Peptide blocking studies (non-quantitative)
• Affinity purification
• Phosphorylation assays
• Protein electrophoresis applications and immunocytochemistry

>95% purity

• ELISA standards and RIA protocols (quantitative)
• Receptor-ligand interaction studies (quantitative)
• In vitro bioassays and in vivo studies.
• Enzyme studies and blocking assays (quantitative)
• NMR studies.
• Mass spectrometry.
• Other quantitative assays.

>98% purity

• SAR Studies
• Clinical trials
• API (Active Pharmaceutical Ingredients)
• Commercial products
• X-Ray crystallography studies
• Other sensitive experiments: Enzyme-substrate studies; receptor-ligand interaction studies; blocking and competition assays

5. What is peptide purity?

Peptide purity is the amount of the target peptide as determined by HPLC at 214 nm, where the peptide bond absorbs. Water and residual salts are not detected UV-spectrophotometrically. Other impurities can also be found in the content includes deletion sequences (shorter peptides lacking one or more amino acids of the target sequence), truncated sequences (generated by capping steps to avoid the formation of deletion peptides), and incompletely deprotected sequences (generated during the synthesis or the final cleavage process).

Peptide purity does not include water and salts in the sample. TFA is resulted from the HPLC purification. The free N terminus and other side chain such as Arg, Lys and His will form trifluoroacetates and thus small amounts of TFA may contaminate the peptides. Peptides are usually delivered as trifluoroacetates (TFA) containing residual water. Even in lyophilized peptides, varying amounts of noncovalently-bound water still exist.

6. What is net peptide content?

The net peptide content is different from the peptide purity. The net peptide content is the percentage of peptides relative to nonpeptidic materials, mostly counterions and moisture. The net peptide content can be determined by amino acid analysis. Please request for quote if you need this service. Usually hydrophilic peptides absorb tiny amount of moisture even under strict lyophilization process. Depending on the purification and lyophilization process, the net peptide content may vary from batch to batch.

7.What salt form should I use?

Peptide is usually delivered as TFA salt. If residual TFA is a problem for your experiment, we recommend other salt forms such as acetate and hydrochloride. These salt forms are usually 20-30% more expensive than the regular TFA salt. This is due to the peptide loss during the salt conversion and more raw materials are needed for this purpose.

8. How are peptides synthesized?

Unlike the natural protein synthesis, peptide is synthesized from the C to N terminus. Peptide synthesis at LifeTein is performed by PeptideSyn technology based on Fmoc or t-Boc chemistry to protect the alpha amino group. The deprotection agent (piperidine for Fmoc, TFA for Boc) frees the alpha amino group in preparation for coupling the next amino acid in the sequence, revealing a new N-terminal amine to which a further amino acid may be activated by one of several reagents and a peptide bond is formed. When the synthesis is finished, peptides are cleaved from the resin and de-protected. Peptides are then precipitated, washed, and then lyophilized.

9. What are your QC standards on my peptide synthesis?

All materials supplied to LifeTein are considered the confidential property of the customer. LifeTein provides free HPLC and MS results with your package. Peptides are purified by reversed phase chromatography. The chromatogram indicates the number and relative amount of by-products. The molecular mass of the peptide is determined by mass spectrometry to confirm that the correct product will be delivered. The MS results also show the masses of the main impurities. Additional analysis can be performed by LifeTein if you request for the net peptide content. The net peptide content can be analyzed by the amino acid analysis or elemental analysis. The methods allow to verify the amino acid composition of the peptides and they are additional confirmation of the peptide identity.All synthetic peptide meeting the purity criteria is sent to the customer. All residual materials are discarded if not meeting purity criteria. These residual materials can be sent to the customer upon request.

10. Can you aliquot my peptides?

At your request, LifeTein can aliquot any of your order into smaller quantities at minimum charge of $3 per tube. It will be much easier for you to do the solubility test on each peptide. LifeTein's special consideration on customers' side shows our support when you need it most. The aliquots will save you time and effort in determining the solubility and save your peptides. Your peptides will have better stability because the repeated thawing/freezing, opening/closing of the container, mishandling, bacterial contamination, peptide oxidation, degradation and aggregation can be prevented.

11. What are APIs, catalog peptides and custom peptide synthesis?

APIs, active pharmaceutical ingredients, are the substances in a drug that are pharmaceutically active such as oxytocin acetate, enfuvirtide acetate and so on. Catalog peptides are commercially available sequences. They are usually produced in bulk at high purity. Custom peptides are usually customized according to customers' requests. For example, specific sequences, modifications, purity or length will be ordered from customers. The turnaround time for most peptides is within 2-3 weeks.

12.What is the minimal quantity for one order?

The minimal quantity to be ordered should be at least 1 mg. There is no upper limit at LifeTein for research and GMP peptides.

13. What is the maximum peptide length that LifeTein can produce?

LifeTein has synthesized a peptide of 120 amino acids in length. Peptides of up to 50 amino acids are routinely synthesized although in general, peptides of more than 30 amino acids can be problematic depending on the hydrophobic residues in the sequences or unusual modifications. Hydrophobicity affects the solubility of the peptide. Those difficult peptides can lead to difficulties in production.

14. What is solid phase synthesis?

Organic reactions are carried out on substrates that are covalently attached to a polymeric resin. The solid phase synthesis is better than the traditional synthesis because the overall reaction is much quicker and can be automated with robots. The synthetic intermediates do not need to be isolated. At each step, reagents are simply washed away.

15. What are resins and linkers?

Resin is the polymeric backbone that substrate is anchored to. Different resins have different swelling properties. For example, Polystyrene swells in non-polar solvents, while polyethylene glycol swells in polar and non-polar solvents. Linker is an intermediate structure between resin and substrate. Different linkers can be used to unmask different functional groups on the substrate.

16. What is a protecting group?

To ensure specific coupling between the required carboxyl and amino groups, the protecting groups should be easily and selectively attached and removed. It is a fragment that bounds to a functional group that blocks its reactivity. Some are acid labile protecting groups such as Boc and tert-Bu ester. Some are base labile protecting groups such as Fmoc and Fm ester. While some are fluoride labile protecting groups such as Tmsec and Tmse ester.

17. What are acetylation and amidation?

Chemically synthesized peptides carry free amino and carboxy termini. The need for N-terminal acetylation or C-terminal amidation has to be stated explicitly when ordering. It is impossible to perform these modifications after synthesis.

N-terminal acetylation and/or C-terminal amidation reduce the overall charge of a peptide so that the solubility may decrease. But the stability of the peptide could be increased because the terminal acetylation/amidation will generate a closer mimic of the native protein. Thus these modifications may increase the biological activity of a peptide.

18. Is a spacer required for fluorescent modification?

Usually, the dyes such as Biotin, FITC can be introduced either N-terminally or C-terminally. We recommend N terminus modification for higher success, shorter turnaround time and easier operation from LifeTein. The reason is that the peptides are synthesized from C terminus to N terminus. So the N terminus modification is the last step in the SPPS protocol and no more specific coupling steps are required. On the contrary, the C terminus modification needs additional steps and is usually more complex.

Most dyes are large aromatic molecules so the incorporation of such bulky molecules may help to avoid interactions between the label and the peptide. This will help to maintain the peptide conformation and its biological activity. It is recommended to include a flexible spacer such as Ahx, which is a 6 carbon linker, to make the fluorescent label more stable. Otherwise FITC could be easily linked to a cysteine thiol moiety or the amino group of lysine at any position.