A peptide that was 99% pure on the day it was made won't stay that way forever. Peptides are chemically delicate, and how a sample is handled and stored has a big effect on its quality. Knowing the main degradation pathways helps researchers interpret analytical data and keep reference material reliable.
Chemical degradation
Oxidation
Methionine, cysteine and tryptophan are especially easy to oxidise. Methionine becomes methionine sulfoxide, which adds 16 Da to the mass and is easy to spot by mass spectrometry. Oxygen, light, trace metals and peroxides in solvents all speed this up.
Deamidation
Asparagine, and more slowly glutamine, can lose its side-chain amide and turn into aspartate or isoaspartate. The sequence Asn–Gly is notoriously prone to this. Deamidation changes the charge and adds about 1 Da, so it can be hard to separate from the intact peptide.
Hydrolysis
Peptide bonds can be cut by water, especially in acid. Bonds next to aspartate, and Asp–Pro bonds in particular, are among the weakest.
Other reactions
- Pyroglutamate formation: an N-terminal glutamine or glutamate can cyclise.
- Diketopiperazine formation: the first two residues can cyclise and break off, especially when proline is in position two.
- Disulfide scrambling: disulfide bonds can swap to the wrong cysteine partners.
- Racemisation: L-amino acids can slowly convert to their D-forms under basic conditions.
Physical instability
Not all degradation breaks bonds. Peptides can also aggregate, sticking together into clusters or highly ordered amyloid-like fibrils, and they can adsorb to the surfaces of glass and plastic containers. Hydrophobic and β-sheet-prone sequences are most at risk. Aggregation can make a sample cloudy, lower the measured concentration and change how it behaves in experiments.
Why freeze-dried powder is preferred
Almost every degradation reaction needs water. That's why peptides are supplied as lyophilised (freeze-dried) powders: removing the water slows chemical change dramatically. A peptide stored dry and cold can keep for years, whereas the same peptide in solution may degrade within days or weeks.
Good laboratory storage practice
| Practice | Why |
|---|---|
| Store lyophilised material at −20 °C or below | Slows all chemical reactions |
| Keep containers sealed and dry, ideally with a desiccant | Peptides absorb moisture from the air |
| Let cold vials reach room temperature before opening | Prevents condensation forming on the powder |
| Protect from light | Light drives oxidation, especially of tryptophan |
| Divide lab solutions into single-use portions | Repeated freeze–thaw cycles promote aggregation |
| Record dates and re-test reference material | Quality can drift over time |
Spotting degradation analytically
Degradation shows up as new peaks in an HPLC chromatogram and as mass shifts in mass spectrometry: +16 Da for oxidation, about +1 Da for deamidation, and fragments from hydrolysis. That's why a certificate of analysis describes a batch on its test date. See How peptide purity is tested.
Related articles
- How laboratories validate analytical methods
- D-amino acids and peptidomimetics
- Why some peptides are hard to make
Sources and further reading
- Manning MC et al. Stability of protein pharmaceuticals: an update. Pharm Res 2010;27:544–575. doi:10.1007/s11095-009-0045-6 · PMID: 20143256
- Mant CT et al. HPLC analysis and purification of peptides. Methods Mol Biol 2007;386:3–55. doi:10.1007/978-1-59745-430-8_1 · PMID: 18604941