This chapter outlines essential guidelines for proper peptide handling, storage, and reconstitution, covering:

• • Best practices for storing lyophilized peptides and maintaining stability
  • Step-by-step guidance for handling and weighing peptide samples
  • Detailed solutions for peptide solubilization based on chemical properties
  • Special considerations for basic, acidic, and hydrophobic peptides
  • Storage recommendations for peptides in solution and shipping conditions

Peptide modification refers to permanent chemical alterations of the molecule, in contrast to protecting groups, which are removed after synthesis to obtain the final product (even in the case of so-called “permanent” protecting groups). Peptides can be modified in various ways, including functionalization of the N- and C-termini, derivatization of reactive groups in the 20 proteinogenic amino acids, and replacing specific amino acids with non-proteinogenic or unusual amino acids.

Stability of lyophilized peptides

If stored under appropriate conditions, peptides are rather stable. Nevertheless, they should not be stored in solution (even sterile and oxygen-free solution) because they may slowly undergo chemical degradation. Frozen solutions may be kept for a few weeks.

For longer storage, peptides should be maintained as the lyophilizate in a tightly closed container at less than −15 °C, although lower temperatures are preferred (e.g., −50 °C or lower for long-term storage). Peptides containing Asn, Gln, Met, Cys, and/or Trp have limited shelf lives. However, they may be shipped at room temperature, and for short-term use, they may be stored in a refrigerator at 4 °C.

Handling of lyophilized peptides

Lyophilized peptides should be weighed out quickly and the container tightly resealed. Because peptides tend to be hygroscopic, the container should be allowed to reach ambient temperature in a desiccator prior to opening and weighing out the peptide. The absorption of moisture from the atmosphere reduces the overall peptide content and may also decrease stability. Please keep in mind to wear a dust respirator when handling larger amounts of peptide lyophilizates.

Solubilization of peptides

The reconstitution of a hydrophobic peptide may turn into an almost insurmountable obstacle in the course of your assay. As the properties of peptides can vary extraordinarily, we are unable to provide a standard protocol for dissolving a peptide. However, we can offer numerous tips and tricks gained from our broad experience in handling these compounds.

The solubility of a peptide is determined mainly by its polarity. The amino acid composition will provide a first indication: before choosing a solvent, the sequence should be studied, and the number of acidic, basic, and neutral residues should be counted. The side-chain functionalities of acidic and basic amino acids will be charged at physiological pH. The nature of the N- and C-terminal functionalities must also be taken into consideration. Neutral amino acids can be divided into two categories: non-polar (i.e., more or less hydrophobic) residues and polar residues. “Dispersed” Pro residues disrupt secondary structures and thus increase the solubility.

Basic peptides

The number of basic amino acids including the N-terminal amino group is larger than the number of acidic amino acids including the C-terminal carboxyl group. They may be dissolved in a small amount of an acidic solvent such as acetic acid or trifluoroacetic acid and then diluted to the desired concentration. However, the safest diluent is PBS at pH 7.0–7.4, provided that a concentration of ≤1 mg/mL is sufficient. If delivered as the trifluoroacetate salt, peptides containing a relatively large proportion of Arg and Lys residues tend to be soluble at neutral pH.

Acidic peptides

The number of acidic amino acids including the C-terminal carboxyl group is larger than the number of basic amino acids including the N-terminal amino group. They may be reconstituted in a small amount of a basic solvent such as 0.1% aqueous ammonia and then diluted with water to the desired concentration. However, as in the case of basic peptides, these may often be solubilized in PBS at pH 7.0–7.4 provided that a concentration of ≤1 mg/mL is sufficient.

Neutral or highly hydrophobic peptides

Peptides containing a high proportion of polar uncharged amino acids and/or hydrophobic amino acids should be dissolved in a small amount of an organic solvent such as DMSO, DMF, acetic acid, acetonitrile, methanol, propanol, or isopropanol and then diluted with water (or buffer) to the desired concentration. Please keep in mind that high concentrations of these solvents are incompatible with biological systems such as cells.

Denaturing agents, such as urea or guanidinium hydrochloride, may be used to solubilize peptides that are susceptible to aggregation. However, as these additives interfere with most biological systems, their application is rather limited.

The reconstitution of a peptide may take time, occasionally up to several hours. Sonication for several minutes in a water bath may be helpful to accelerate the dissolution of larger particles. However, excessive warming of the sample should be avoided.

Please note that peptides containing Trp, Met, or Cys residues require special care to avoid oxidation. Oxygen-free water/buffers or reducing agents such as 1,4-dithio-dl-threitol (DTT) have to be used. In addition, peptides containing free Cys residues should be dissolved in carefully degassed acidic buffers, as the thiol moieties will be rapidly oxidized to the disulfides at pH > 7.

Storing peptides in solution

As discussed above, peptides should be dissolved in an appropriate buffer. For storage, peptide solutions should be aliquoted and kept frozen below −15 °C. The long-term storage of peptide solutions is not recommended, especially when the peptide contains Asn, Gln, Cys, Met, or Trp residues.

Delivery times for our peptides

Bachem prides itself on efficient delivery, ensuring that most catalogue and custom peptide orders arrive within 1–2 working days. Products are typically shipped at ambient temperature, with recommended long-term storage conditions clearly noted on the label. Orders can be dispatched via mail, airmail, or premium services like FedEx and UPS, tailored to your location and requirements. For customers in the US and Canada, standard overnight or second-day delivery options are available. Find out more about our delivery times in our knowledge center.

This concludes the final chapter of our peptide guide. This comprehensive guide has walked you through the crucial aspects of peptide science and manufacturing, from basic concepts to advanced production methods. For those that are new to peptide chemistry or looking to deepen their understanding, we’ve covered everything from the fundamental building blocks of amino acids to sophisticated synthesis techniques like SPPS and Molecular Hiving.