Peptide Synthesis: Handling and Storage of Synthetic Peptides

1. How should I store and handle my synthesized peptides?
2. How should I dissolve peptides?
3. Can I predict whether a peptide will be soluble?
4. How do I choose the best level of peptide purity for my research?
5. What is peptide purity?
6. What is net peptide content?
7. What salt form should I use?
8. How are peptides synthesized?
9. What are your QC standards on my peptide synthesis?
10. Can you aliquot my peptides?
11. What are APIs, catalog peptides and custom peptide synthesis?
12. What is the minimum quantity for one order?
13. What is the maximum peptide length that LifeTein^® can produce?
14. What is solid-phase synthesis?
15. What are resins and linkers?
16. What is a protecting group?
17. What are acetylation and amidation?
18. Is a spacer required for fluorescent modification?

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

Peptides shipped at room temperature are highly stable at lyophilized form in sealed bags (Why in sealed bags?). Peptides should not be kept in solution for long periods.

Peptide storage guidelines: For long-term storage, peptides should be stored in lyophilized form at -20°C or preferably at -80°C with desiccant in sealed containers in order to minimize peptide degradation. Under these conditions, peptides can be stored for up to several years. This type of storage prevents bacterial degradation, oxidation, and the formation of secondary structures.

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

Weighing peptides: Weigh out your needed quantity of peptide quickly and store all unused peptide at -20°C or below. Sequences containing cysteine, methionine, tryptophan, asparagine, glutamine, and N-terminal glutamic acid will have a shorter shelf lives 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 and neutral peptides containing large numbers of hydrophobic or polar uncharged amino acids should be dissolved in a small amounts of organic solvent such as DMSO, DMF, acetic acid, acetonitril, methanol, propanol or isopropanol and then diluted with water. DMSO should not be used with peptides containing methionine or free cysteine because it may oxidize the side-chain.

Test a portion of the synthesized peptide before dissolving the rest of the sample. You may need to test several different solvents until you find an appropriate one. Sonication enhances solubilization.

1. Assign a value of -1 to each acidic residue. The acidic residues are Asp (D), Glu (E), and the C-terminal -COOH. Assign a value of +1 to each basic residue. The basic residues are Arg (R), Lys (K), His (H), and the N-terminal -NH2. Then calculate the overall charge of the peptide.
2. If the overall charge of the peptide is positive, the peptide is basic. Try to dissolve the peptide in distilled water if possible. If it fails to dissolve in water, then try to dissolve the peptide in a small amount of 10-25% acetic acid. If this fails, add TFA (10-50 µl) to solubilize the peptide and dilute it to your desired concentration.
3. If the overall charge of the peptide is negative, the peptide is acidic. Acidic peptides may be soluble in PBS (pH7.4). If this fails, add a small amount of basic solvent such as 0.1M ammonium bicarbonate to dissolve the peptide and add water to the desired concentration. Peptides containing free cysteines should be dissolved in degassed acidic buffers. The thiol moieties will be rapidly oxidized to disulfides at pH>7.
4. If the overall charge of the peptide is zero, the peptide is neutral. Neutral peptides usually dissolve in organic solvents. First, try to add a small amount of acetonitrile, methanol, or isopropanol. For very hydrophobic peptides, try to dissolve the peptide in a small amount of DMSO, and then dilute the solution with water to the desired concentration. For Cys-containing peptides, use DMF instead of DMSO. For peptides that tend to aggregate, add 6 M guanidine.HCl or 8 M urea, and then proceed with the necessary dilutions.

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 a solution, freeze-thaw cycles should be avoided by freezing individual aliquots.

Positively charged residues: K, R, H, N-terminus
Negatively charged residues: D, E, C-terminus
Hydrophobic uncharged residues: F, I, L, M, V, W, Y
Uncharged residues: G, A, S, T, C, N, Q, P, acetyl, amide

Examples:
RKDEFILGASRHD: (+5) + (-4) = +1 This is considered a basic peptide. See step #2 above.
EKDEFILGASEHR: (+4) + (-5) = -1 This is considered an acidic peptide. See step #3 above.
AKDEFILGASEHR: (+4) + (-4) = 0 This is considered a neutral peptide. See step #4 above.

3. Can I predict whether a peptide will be soluble?

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

Certain basic characteristics can be used to predict solubility:

• Peptides that are shorter than 5 amino acids are usually soluble in aqueous solutions. If the entire sequence consists of hydrophobic amino acids, it will have limited solubility or may even be completely insoluble.
• Hydrophilic peptides containing >25% charged residues (E, D, K, R, and H) and <25% hydrophobic amino acids are usually soluble in aqueous solutions.
• Hydrophobic peptides containing 50% or more hydrophobic residues may be insoluble or only partly soluble in aqueous solutions. It is better to dissolve these peptides in organic solvents such as dimethylsulfoxide (DMSO) if they do not contain C, W or M, dimethylformamide (DMF), acetonitrile, isopropyl alcohol, ethanol, acetic acid, 4–8 M guanidine hydrochloride (GdnHCl), or urea prior to a careful dilution in aqueous solution.
• Hydrophobic peptides containing >75% hydrophobic residues generally do not dissolve in aqueous solutions.  Very strong solvents such as TFA and formic acid are required for the initial solubilization. The peptide may precipitate when added to an aqueous buffered solution. High concentration of organic solvent or denaturant may be required to dissolve these peptides.
• Peptides containing a very high proportion (>75%) of D, E, H, K, N, Q, R, S, T, or Y are capable of building intermolecular hydrogen bonds (cross-links) and can thus form gels in concentrated aqueous solutions. These peptides should be dissolved in organic solvents. The initial solvent of choice should be compatible with the experiment. After dissolving the peptides in organic solvent, slowly add (dropwise) the solution to a stirred aqueous buffer solution. If the resulting peptide solution begin to show turbidity, you have reached the limit of solubility.

4. How do I choose the best level of peptide purity for my research?

Crude peptides are not recommended for biological assays. Crude peptides may contain large amounts of non-peptide impurities such as residual solvents, scavengers from cleavage, TFA and other truncated peptides. TFA cannot be totally removed. Peptides are 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 that takes place during the salt conversion and the greater amounts of raw materials required.

LifeTein^® recommends the following levels of peptide purity for various projects:

>70% purity

• Peptide arrays
• Antigens for antibody production
• Competitive elution chromatography
• ELISA standards 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
• APIs (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 by UV spectrophotometer. Other impurities that can be found in the content include 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 any water or salts in the sample. TFA results from HPLC purification. The free N terminus and other side chains such as Arg, Lys, and His form trifluoroacetates and this allows small amounts of TFA to contaminate the peptides. Peptides are usually delivered as trifluoroacetates containing residual water. Even in lyophilized peptides, varying amounts of noncovalently bound water still exist.

What are other substances (impurities) in the peptides?

The impurities in non-purified peptides are both peptides and non-peptides, the impurities in purified peptides are mostly peptides with modified sequences, except for TFA salt.

1. Shorter peptides lacking one or more amino acids of the target sequence
2. Generated by capping steps to avoid the formation of deletion peptides
3. Generated during the synthesis or the final cleavage process
4. Reattachment of protecting groups at other locations on the peptide

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 place a request for a quote if you require this service. Usually, hydrophilic peptides absorb tiny amounts of moisture even under strict lyophilization conditions. Net peptide content may vary from batch to batch depending on the purification and lyophilization processes.

7.What salt form should I use?

Peptides are usually delivered as TFA salts. If residual TFA would be problematic 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 because of the peptide loss that takes place during the salt conversion and the greater amounts of raw materials required.

8. How are peptides synthesized?

Unlike the natural protein synthesis, peptides are synthesized from the C to N terminus. At LifeTein^®, peptide synthesis is performed using 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. This reveals a new N-terminal amine to which the next amino acid may be activated by one of several reagents, forming a peptide bond. When the synthesis is complete, peptides are cleaved from the resin and de-protected. Peptides are then precipitated, washed, and lyophilized.

9. What are your QC standards for 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 reverse-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 is being delivered. MS results also show the masses of the main impurities. Additional analysis revealing net peptide content can be performed upon request. Net peptide content is indicated by either amino acid analysis or elemental analysis. These methods allow the verification of the amino acid composition of the peptides. They serve as additional means of confirmation of peptide identity. All synthetic peptides meeting the customer's purity criteria are sent. All residual materials, such as peptides not meeting the customer's purity criteria are discarded. These residual materials can be sent to the customer upon request.

10. Can you aliquot my peptides?

Upon request, LifeTein^® can aliquot part or all of your order into smaller quantities for a minimum fee of $3 per tube. Aliquoted products are more expensive but may save you time, effort and money during determination of peptide solubility. Your peptides will also be more stable because they will not be exposed to as many freeze-thaw cycles, as many openings and closings of the container, mishandling, or bacterial contamination. Peptide oxidation, degradation, and aggregation are less prevalent in aliquoted samples.

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

APIs (active pharmaceutical ingredients) are the substances in drugs 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 levels of purity. These peptides are usually customized to customers' specific requests. For example, specific sequences, modifications, purity levels, or lengths may be required by the customer. The turnaround time for most API peptides is 2-3 weeks.

12.What is the minimum quantity for one order?

The minimumal quantity to be ordered should be at leastis 1 mg. At LifeTein^®, There there is no maximum upper limit at LifeTein for research and or 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 50 amino acids are synthesized routinely.

14. What is solid-phase synthesis?

Organic reactions are carried out on substrates covalently attached to a polymeric resin. Solid-phase synthesis can be better than the traditional synthesis because the overall reaction takes place much more quickly, the process can be automated with robots, and synthetic intermediates do not need to be isolated because reagents are washed away during each step.

15. What are resins and linkers?

Resin is the polymeric backbone to which substrates are anchored. Different resins have different properties. For example, polystyrene swells in non-polar solvents, while polyethylene glycol swells in polar and non-polar solvents. Linkers are intermediate structures that attack the resin to the substrate. Different linkers can be used to unmask different functional groups on the substrate.

16. What is a protecting group?

Protecting groups are fragments that binds to functional groups and blocks their 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. Some others are fluoride-labile protecting groups such as Tmsec and Tmse ester. To ensure specific coupling between the required carboxyl and amino groups, the protecting groups should be easy to attach and remove without changing the rest of the peptide.

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 must be stated explicitly during ordering. It is impossible to perform these modifications after synthesis has been completed.

N-terminal acetylation and C-terminal amidation reduce the overall charge of a peptide and decrease solubility. However the stability of the peptide usually increases because the terminal acetylation and amidation allow the peptide to mimic the native protein more closely. In this way, these modifications may increase the peptide's biological activity.

18. Is a spacer required for fluorescent modification?

Usually, dyes such as biotin and FITC can be introduced either N-terminally or C-terminally. We recommend N-terminus modification for its higher success rate, shorter turnaround time, and ease of operation. Peptides are synthesized from the C-terminus to the N-terminus. N-terminus modification is the last step in the SPPS protocol. No more specific coupling steps are required. In contrast, the C-terminus modification requires additional steps and is usually more complex.

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