Peptide Stability, Storage and Solubility

Storing lyophilised peptides

Peptides are usually supplied as a freeze-dried (lyophilised) powder, often as a TFA or acetate salt. In this form water is largely absent, and most chemical reactions that degrade peptides in solution proceed extremely slowly. The main enemies of a dry peptide are moisture, heat, light and oxygen.

• Temperature. Store at −20 °C for routine use and at −80 °C for long-term storage. Many peptides tolerate room temperature for days to weeks, which is why they can be shipped without cooling, but cold storage slows every degradation pathway.
• Moisture. Lyophilised peptides are hygroscopic, especially as salts and when they contain many charged residues. Keep vials tightly closed, ideally in a sealed container with desiccant.
• Warm before opening. A vial taken straight from the freezer and opened at once draws in humid air, and water condenses on the cold powder. Let it reach room temperature, closed, before opening, typically 20–30 minutes.
• Light and air. Peptides containing Trp, Tyr, Met or Cys are sensitive to light and oxygen. Amber vials and flushing the headspace with argon or nitrogen before resealing help for long-term storage.

Weighing out small portions repeatedly from one stock vial exposes the whole batch to moisture each time. For peptides used over months, it is better to dissolve the whole batch once and store aliquots, or to divide the powder into several vials at the start.

Choosing a solvent

No single solvent suits every peptide. The best starting point is the peptide's net charge at neutral pH, which the MW calculator reports directly and the isoelectric point guide explains. Solubility is lowest near the pI, so the aim is to dissolve at a pH well away from it.

Some general rules help in every case:

• Test a small amount first before committing the whole batch to a solvent.
• Dissolve at high concentration, then dilute. Adding the concentrated peptide solution to the final buffer usually works better than adding buffer to dry powder.
• Brief sonication in a water bath can help break up particles. Avoid prolonged sonication, which heats the sample.
• A clear solution is not proof of full dissolution. Fine aggregates can scatter little light. Centrifuge before use and, if possible, check the concentration of the supernatant.

Why avoid DMSO for Cys- and Met-containing peptides? DMSO is a mild oxidant: it is used deliberately to form disulfide bonds, and it can oxidise methionine. Basic conditions are avoided for free cysteines for a related reason: the thiolate anion, which forms above about pH 8, is the species that reacts with oxygen and with other thiols.

Storing peptide solutions

In solution, the shelf life of a peptide shortens from years to weeks or days, depending on the sequence and conditions. A few practices extend it considerably:

• Aliquot and freeze. Divide the stock into single-use portions and store them at −20 °C or −80 °C. Each freeze–thaw cycle can promote aggregation and concentrates solutes in the unfrozen liquid, which speeds up reactions.
• Choose the pH deliberately. Many peptides are most stable around pH 5–6: deamidation accelerates at higher pH, while acid-catalysed cleavage and Asp isomerisation increase at low pH.
• Use sterile, clean buffers. Microbial growth and trace metal ions, which catalyse oxidation, are common causes of unexplained degradation. Filtering through a 0.2 µm filter removes microorganisms.
• Degas buffers for peptides with Cys, Met or Trp, and avoid unnecessary headspace in storage tubes.

Chemical degradation pathways

Most chemical instability can be predicted from the sequence. The table summarises the main reactions and the mass change each one produces, which is how they are usually detected by mass spectrometry:

Oxidation

Methionine is the residue most easily oxidised. Its sulfoxide adds one oxygen atom and often changes biological activity and HPLC retention; it can be reversed chemically, but that is rarely convenient. Free cysteines form intermolecular disulfides, producing dimers and oligomers that appear at roughly twice the mass. If a peptide is meant to have free thiols, reducing agents such as DTT or TCEP can keep them reduced, but these interfere with some assays.

Deamidation and isomerisation

Asparagine deamidates through a cyclic succinimide intermediate that forms when the backbone nitrogen of the next residue attacks the Asn side chain. The succinimide then opens to give a mixture of normal aspartate and isoaspartate, in which the chain continues through the side chain rather than the α-carboxyl; isoaspartate usually predominates, typically by about three to one. The reaction is fastest when the next residue is small and flexible, above all glycine, and its half-life at neutral pH and 37 °C can be as short as a day for Asn-Gly sequences.

Aspartate forms the same succinimide more slowly, through loss of water, and so isomerises without any change in mass. Isoaspartate is therefore easy to miss by MS and usually shows up as a new, closely eluting HPLC peak.

Cyclisation at the N-terminus

An N-terminal glutamine readily cyclises into pyroglutamate, losing ammonia. This removes the free N-terminal amine, which changes the charge and makes the peptide resistant to Edman sequencing and to aminopeptidases. Many natural hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone, carry pyroglutamate by design; in a synthetic peptide with an N-terminal Gln it is usually an unwanted by-product.

Physical instability

Aggregation

Peptides with long hydrophobic stretches or a tendency to form β-sheets can assemble into aggregates ranging from small soluble oligomers to amyloid-like fibrils. Glucagon, for example, forms fibrils in concentrated acidic solution, and amyloid-β peptides aggregate readily. Aggregation depends on concentration, pH, ionic strength, temperature and agitation, and it is often irreversible. A strongly positive hydropathy profile and a pH close to the pI are the main warning signs.

Adsorption to surfaces

At low concentrations, a large fraction of a peptide can be lost by sticking to the walls of tubes, pipette tips and vials. Hydrophobic and strongly cationic peptides are most affected, and losses become significant below roughly 1 µM. Low-binding polypropylene tubes, a small proportion of organic solvent, or a carrier protein such as BSA in the buffer reduce the problem. Diluting from a concentrated stock just before use also helps.

A pre-use checklist from the sequence

Reading the sequence before opening the vial answers most practical questions:

1. Net charge at pH 7 decides the first solvent to try.
2. Cys present? Avoid DMSO and basic pH unless disulfide formation is intended; consider degassed buffers.
3. Met or Trp present? Protect from light and oxygen; expect +16 Da peaks in old samples.
4. Asn-Gly, Asn-Ser or Asp-Gly present? Keep solutions slightly acidic, cold, and short-lived.
5. Asp-Pro present? Avoid prolonged exposure to strong acid.
6. N-terminal Gln? Expect gradual pyroglutamate formation in solution.
7. Long hydrophobic stretch or high GRAVY? Dissolve in organic solvent first, work at low concentration, and use low-binding plastics.

Frequently asked questions

References

• Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS (2010) Stability of protein pharmaceuticals: an update. Pharmaceutical Research 27:544–575.
• Geiger T, Clarke S (1987) Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Journal of Biological Chemistry 262:785–794.
• Robinson NE, Robinson AB (2001) Molecular clocks. PNAS 98:944–949.

Related guides

Browse all 27 guides →